Berberine salts, ursodeoxycholic salts and combinations, methods of preparation and application thereof

ABSTRACT

The invention provides various novel compositions of berberine in combination with pharmacologically active organic acids, and related methods of their use in treating various diseases or disorders. The invention further provides various novel compounds prepared from berberine and pharmacologically active organic acids and prepared from ursodeoxycholic acid and pharmacologically active organic bases, and pharmaceutical compositions thereof, and methods of their preparation and therapeutic use in treating and/or preventing various diseases or disorders. The compounds and pharmaceutical compositions of the invention can be utilized to treat various diseases or disorders, such as diabetes, diabetic complications, dyslipidemia, hyperlipidemia, obesity, metabolic syndromes, pre-diabetes, atherosclerosis, heart diseases, neurodegenerative diseases, sarcopenia, muscle atrophy, inflammation, cancer and liver diseases and conditions such as fatty liver, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, cholestatic liver diseases or graft-versus-host disease of the liver. The compounds of this invention are also useful in improving liver functions in chronic viral associated liver diseases and alcohol-related liver diseases.

PRIORITY CLAIMS AND RELATED APPLICATIONS

This application is the U.S. national phase of and claims the benefit ofpriority to PCT/CN2015/085350, filed Jul. 28, 2015, which claims thebenefit of priority from U.S. Provisional Application Nos. 62/030,140and 62/030,147, each filed Jul. 29, 2014, and U.S. ProvisionalApplication No. 62/128,077, filed Mar. 4, 2015, the entire content ofeach of which is incorporated herein by reference in its entirety.

TECHNICAL FIELD OF THE INVENTION

The invention generally relates to novel therapeutic compounds,pharmaceutical compositions, and methods of preparation and therapeuticuse thereof.

In particular, the invention relates to novel compositions of berberinein combination with pharmacologically active organic acids, and methodsof their use. In particular, the invention also relates to novel saltsof berberine and organic acids and novel salts of ursodeoxycholic acidand organic bases, pharmaceutical compositions thereof, methods of theiruse. The compounds and pharmaceutical compositions of the invention areuseful in treating and/or preventing various diseases or disorders,including metabolic diseases or disorders such as pre-diabetes,diabetes, diabetic complications, dyslipidemia, dyslipidemia instatin-intolerance patients, hyperlipidemia, hypercholesterolemia,hypertriglyceridemia, diabetic dyslipidemia, or obesity. Additionally,the compounds and pharmaceutical compositions of the invention areuseful in treating and/or preventing atherosclerosis, heart diseases,neurodegenerative diseases, sarcopenia, muscle atrophy, inflammation,cancers, as well as various liver diseases or disorders, such as fattyliver, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis,cholestatic liver diseases or graft-versus-host disease of the liver.Furthermore, the compounds and pharmaceutical compositions of theinvention are useful in improving liver functions in chronic viralassociated liver diseases and alcohol-related liver diseases.

BACKGROUND OF THE INVENTION

Diabetes mellitus is a disorder of metabolism. It has become pandemicwith an estimate of over 300 million people worldwide living withdiabetes today. Without effective prevention, this number will grow upto 500 million by 2030. There are three main types of diabetes: type 1diabetes, type 2 diabetes, and gestational diabetes. Among them, type 2diabetes, is the most common form of diabetes accounting for 90-95% ofcases. Type 2 diabetes is characterized by impaired insulin secretion,increased hepatic glucose production, and decreased response ofperipheral tissues to insulin, i.e., insulin resistance. Manytherapeutic treatments are available for the management of type 2diabetes, but they are often accompanied by various side effects. Anoptimal therapy should be safe and include early initiation ofcombination drugs with complimentary mechanisms of action.

Despite persistent efforts and meaningful progress over the past decadesin the understanding and management of diabetes, people with diabetescontinue to have an increased risk of, and many do suffer from, a numberof serious complications inflicting the heart and blood vessels, eyes,kidneys, and nerves due to high blood glucose, high cholesterol, andhigh blood pressure. Cardiovascular diseases are the most common causeof death in people with diabetes. Diabetic nephropathy caused by damagesto small blood vessels in the kidney leads to decreased kidney functionor kidney failure altogether. Diabetic neuropathy is caused by damagesto the nerves throughout the body when blood glucose level and bloodpressure are too high. Most people with diabetes develop diabeticretinopathy causing reduced vision or blindness. Consistently highlevels of blood glucose, together with high blood pressure and highcholesterol, are the main causes of diabetic retinopathy. Despite thedevelopment of a number of anti-diabetic agents, there are significantunmet needs for therapeutics that can be used effectively for thetreatment and management of diabetic complications.

Metabolic syndrome is a term that refers to a group of risk factors thatoccur together (e.g., abdominal (central) obesity, elevated bloodpressure, elevated fasting plasma glucose, high serum triglycerides, andlow high-density cholesterol (HDL) levels). Metabolic syndrome has beendemonstrated to increase the risk of developing cardiovascular diseases,particularly heart failure, and diabetes. Studies have estimated thatthe prevalence of metabolic syndromes in the US to be around 34% in theadult population. While therapeutics are available, the first linetreatment is change of lifestyle. High-dose statins, recommended toreduce cardiovascular risks, have been linked to higher progression todiabetes, especially in patients with metabolic syndrome.

Dyslipidemia is a disorder of lipoprotein metabolism, includinglipoprotein overproduction (hyperlipidemia) or deficiency. Dyslipidemiasmay be manifested by elevation of the total cholesterol, the “bad”low-density lipoprotein cholesterol and the triglyceride concentrations,and a decrease in the “good” high-density lipoprotein cholesterolconcentration in the blood. Dyslipidemia comes under consideration inmany situations including diabetes, a common cause of dyslipidemia.Hyperlipidemia is elevation of plasma cholesterol(hypercholesterolemia), triglycerides (hypertriglyceridemia), or both,or a low high-density lipoprotein level that contributes to thedevelopment of atherosclerosis. Causes may be primary (genetic) orsecondary. Diagnosis is by measuring plasma levels of total cholesterol,TGs, and individual lipoproteins. Treatment involves dietary changes,exercise, and lipid-lowering drugs.

Cardiovascular disease (CV), often used interchangeably with the term‘heart disease’, refers to a range of conditions that affect the heartsuch as coronary artery disease, arrhythmias, congestive heart failure,cerebrovascular disease etc. Many forms of CV can be prevented ortreated with healthy lifestyle choices, by controlling conditions suchas atherosclerosis, high blood pressure, diabetes or obesity with averity of medicines such as antiplatelet drugs, anticoagulants,digitalis, angiotensin converting enzyme (ACE) inhibitors, betablockers, and LDL cholesterol-lowering agents etc. Due to thecomorbidity, patients often need to take multiple medicines, and itwould be desirable if one pill can target multiple abnormalities.

With demonstrated ability to prevent cardiovascular disease, statins areamong one of the most widely prescribed medications. Although statinsare generally well tolerated, statin intolerance occurs in some patientsand requires careful consideration. In addition, patients are sometimesconcerned about the potential risk of statins causing diabetes mellitus,cancer, and memory loss and often question whether they should continuewith their medication. For statin-intolerant patients, non-statinLDL-C-lowering drugs can be used; however, till the PCSK9 inhibitors areapproved, none of the approved drugs has been nearly as effective asstatins. Developing alternative and effective therapeutics for thesepatients is much needed.

Neurodegenerative disease is an umbrella term for a range of conditionsthat primarily affect the neurons in the human brain. Neurons are thebuilding blocks of the nervous system that includes the brain and spinalcord. Neurons normally don't reproduce or replace themselves when theybecome damaged or die. Examples of neurodegenerative diseases includeParkinson's, Alzheimer's, and Huntington's disease. Neurodegenerativediseases are incurable and debilitating conditions that result inprogressive degeneration and/or death of nerve cells. The unmet medicalneeds for neurodegenerative diseases desperately call for thedevelopment of effective therapeutics.

Muscle atrophy is a decrease in the mass of the muscle, which caninvolve a partial or complete wasting away of muscle. Muscle atrophyoccurs due to changes in the balance between protein synthesis anddegradation. Muscle atrophy can significantly affect a patient's qualityof life as the patient becomes unable to perform certain tasks or risksaccidents (e.g., falling). Muscle atrophy is associated with aging andcan be a serious consequence of different diseases, including cancer,AIDS, and diabetes. Comparing to non-diabetic older adults, elderly withtype 2 diabetes have lower skeletal muscle strength, and are oftenassociated with excessive loss of skeletal muscle mass. Currently, thereare no drugs approved for the treatment of skeletal muscle atrophy.

Sarcopenia is characterized first by a muscle atrophy, along with areduction in muscle tissue quality, characterized by such factors asreplacement of muscle fibers with fat, an increase in fibrosis, changesin muscle metabolism, oxidative stress, and degeneration of theneuromuscular junction and leading to progressive loss of musclefunction and frailty, currently, there is no approved therapeutics forsarcopenia.

Cancer is a group of diseases involving abnormal cell growth with thepotential to invade or spread to other parts of the body. In 2012, about14 million new cases of cancer occurred globally. The most common typesof cancer include lung cancer, prostate cancer, colorectal cancer andstomach cancer for men, and breast cancer, colorectal cancer, lungcancer and cervical cancer for women. While many treatment options forcancer exist, including surgery, chemotherapy, radiation therapy,hormonal therapy, targeted therapy and palliative care, cancer remains atop health threat and is responsible for about 15% of all human deaths.

Fatty liver is a reversible condition wherein large vacuoles oftriglyceride fat accumulate in liver cells via the process of steatosis.Despite having multiple causes, fatty liver can be considered a singledisease that occurs worldwide in those with excessive alcohol intake andthe obese. Non-alcoholic fatty liver disease (NAFLD) is a form of fattyliver diseases that occurs when excessive fat is deposited in the liverof patients without excessive alcohol intake. NAFLD is generallyrecognized to be associated with metabolic syndrome such as insulinresistance, hypertension and obesity. NAFLD affects about a third of theadult population in developed countries. Non-alcoholic steatohepatitis(NASH) is the most extreme form of NAFLD with chronic inflammation thatcan lead to progressive fibrosis (scarring), cirrhosis, and eventualliver failure and death. NASH resembles alcoholic liver disease, butoccurs in people who drink little or no alcohol. A major feature of NASHis fat in the liver, along with inflammation and damage. Most peoplewith NASH, an often “silent” liver disease, feel well and are not awarethat they have a liver problem. Nevertheless, NASH can be severe and canlead to cirrhosis, when the liver is permanently damaged and scarred andno longer work properly.

Currently, there are no drugs approved for the treatment of NASH, whichoccurs in about a quarter of patients with NAFLD. The current standardof care for NASH involves weight loss and increased physical activities.NASH affects 2-5% of Americans and is becoming more common, possiblybecause of the greater number of Americans with obesity. In the past 10years, the rate of obesity has doubled in adults and tripled inchildren.

The therapeutics and methods currently available for the management ofdiseases or disorders such as diabetes, diabetic complications,dyslipidemia, obesity, metabolic syndromes, pre-diabetes, Heartdiseases, neurodegenerative diseases, NAFLD, NASH, muscle atrophy,inflammation and cancers are suboptimal. There remains an ongoing andurgent need for novel and improved therapeutics and methods to treatsuch diseases or disorders.

SUMMARY OF THE INVENTION

The invention is based in part on various novel compositions ofberberine in combination with pharmacologically active organic acids,and related methods of their use in treating and/or preventing variousdiseases or disorders.

The invention is also based in part on various novel compounds preparedfrom berberine and pharmacologically active organic acids, various novelcompounds prepared from ursodeoxycholic acid and pharmacologicallyactive organic bases, and pharmaceutical compositions thereof, andmethods of their preparation and therapeutic use in treating and/orpreventing various diseases or disorders.

The compounds and pharmaceutical compositions of the invention can beutilized to treat various diseases or disorders, such as diabetes,diabetic complications, dyslipidemia, dyslipidemia in statin-intolerancepatients, hyperlipidemia, hypercholesterolemia, hypertriglyceridemia,diabetic dyslipidemia, obesity, metabolic syndromes, pre-diabetes, heartdiseases, neurodegenerative diseases, sarcopenia, muscle atrophy,inflammation, and cancers as well as various liver diseases ordisorders, such as fatty liver, non-alcoholic fatty liver disease,non-alcoholic steatohepatitis, cholestatic liver diseases orgraft-versus-host disease of the liver. The compounds of this inventionare also useful in improving liver functions in chronic viral associatedliver diseases and alcohol-related liver diseases.

In one aspect, the invention generally relates to a compositioncomprising: (a) berberine or a derivative or analog thereof; (b) one ormore pharmacologically active organic acids; and (c) optionally apharmaceutically acceptable excipient, carrier, or diluent. Theberberine and the pharmacologically active organic acid(s) are presentin amounts that, when administered to a subject, are sufficient totreat, prevent, or reduce one or more diseases or disorders selectedfrom metabolic disorders, heart diseases, neurodegenerative diseases,muscle atrophy, inflammation, and cancer, or a related disease ordisorder thereof in a mammal, including a human.

In another aspect, the invention generally relates to a method fortreating, reducing, or preventing a metabolic disorder. The methodincludes administering to a subject in need thereof a pharmaceuticalcomposition, which includes: (a) berberine or a derivative or analogthereof; (b) one or more pharmacologically active organic acids, in atherapeutically effective amount, and (c) optionally a pharmaceuticallyacceptable excipient, carrier, or diluent.

In yet another aspect, the invention generally relates to a kit thatincludes: (i) a first agent of berberine or a derivative or analogthereof; (ii) one or more second agent(s) selected from R-(+)-α-lipoicacid, hydroxycitric acid, eicosapentaenoic acid, docosahexaenoic acid,docosapentaenoic acid, ursolic acid, corosolic acid, cinnamic acid,cholic acid, obeticholic acid, ursodeoxycholic acid, oleanolic acid,salicylic acid, betulinic acid, chlorogenic acid, caffeic acid, bassicacid, acetyl L-carnitine, S-allyl cysteine sulphoxide, S-methyl cysteinesulfoxide, pantothenic acid, ascorbic acid, retinoic acid, rhein,nicotinic acid, biotin, and other organic acid that is generallyrecognized pharmacologically active for one or more diseases ordisorders selected from metabolic disorders, heart diseases,neurodegenerative diseases, muscle atrophy, inflammation, and cancer, ora related disease or disorder thereof in a mammal, including a human bythose of skill in the art. The first and second agents can be either bea purified active pharmaceutical ingredient or as an active ingredientfrom natural extract, for examples: bile acids (cholic acid, deoxycholicacid etc.), rhubarb extracts (rhein), cinnamon extract (cinnamic acid),banaba extract (corosolic acid) etc.; and (iii) instructions foradministering the combined agents to a patient having or at risk ofhaving one or more diseases or disorders selected from metabolicdisorders, heart diseases, neurodegenerative diseases, muscle atrophy,and cancer.

In yet another aspect, the invention generally relates to an acid-baseaddition salt in substantially pure form, having the formula of:(X⁺)_(m)(U⁻)_(n)  (I)wherein

(a) U⁻ is an anionic moiety of ursodeoxycholic acid or a derivative oranalog thereof;

(b) X⁺ is a cationic moiety of a pharmacologically active organic base;and

(c) m and n are integers independently selected from 1, 2, 3, 4, 5 and 6so as to arrive at a charge neutral salt.

In yet another aspect, the invention generally relates to apharmaceutical composition comprising an amount of an acid-base additionsalt having the formula of:(X⁺)_(m)(U⁻)_(n)  (I)wherein

(a) U⁻ is an anionic moiety of ursodeoxycholic acid or a derivative oranalog thereof;

(b) X⁺ is a cationic moiety of a pharmacologically active organic base;and

(c) m and n are integers independently selected from 1, 2, 3, 4, 5 and 6so as to arrive at a charge neutral salt,

effective to treat, prevent, or reduce one or more diseases or disordersselected from fatty liver, NAFLD and NASH, cholestatic liver diseases,graft-versus-host disease of the liver, chronic viral associated liverdiseases, alcohol-related liver diseases, metabolic diseases ordisorders such as pre-diabetes, diabetes, diabetic dyslipidemia,dyslipidemia in statin-intolerance patients, hyperlipidemia, obesity ora related disease or disorder thereof in a mammal, including a human,and a pharmaceutically acceptable excipient, carrier, or diluent.

In yet another aspect, the invention generally relates to a method fortreating, reducing, or preventing a disease or disorder. The methodincludes administering to a subject in need thereof a pharmaceuticalcomposition comprising an amount of an acid-base addition salt havingthe formula of:(X⁺)_(m)(U⁻)_(n)  (I)wherein

(a) U⁻ is an anionic moiety of ursodeoxycholic acid or a derivative oranalog thereof;

(b) X⁺ is a cationic moiety of a pharmacologically active organic base;and

(c) m and n are integers independently selected from 1, 2, 3, 4, 5 and 6so as to arrive at a charge neutral salt,

effective to treat, prevent, or reduce one or more diseases or disordersselected from fatty liver, NAFLD and NASH, cholestatic liver diseases,graft-versus-host disease of the liver, chronic viral associated liverdiseases, alcohol-related liver diseases, metabolic diseases ordisorders such as pre-diabetes, diabetes, diabetic dyslipidemia,dyslipidemia in statin-intolerance patients, hyperlipidemia, obesity ora related disease or disorder thereof in a mammal, including a human,and a pharmaceutically acceptable excipient, carrier, or diluent.

In yet another aspect, the invention generally relates to an acid-baseaddition salt in substantially pure form, having the formula of:(B⁺)_(m)(Y⁻)_(n)  (II)wherein

(a) B⁺ is a cationic moiety of berberine or a derivative or analogthereof;

(b) Y⁻ is an anionic moiety of a pharmacologically active organic acid;and

(c) m and n are integers independently selected from 1, 2, 3, 4, 5 and 6so as to arrive at a charge neutral salt.

In yet another aspect, the invention generally relates to apharmaceutical composition comprising an amount of an acid-base additionsalt having the formula of:(B⁺)_(m)(Y⁻)_(n)  (II)wherein

(a) B⁺ is a cationic moiety of berberine or a derivative or analogthereof;

(b) Y⁻ is an anionic moiety of a pharmacologically active organic acid;and

(c) m and n are integers independently selected from 1, 2, 3, 4, 5 and 6so as to arrive at a charge neutral salt,

effective to treat, prevent, or reduce one or more diseases or disordersselected from metabolic disorders, heart diseases, neurodegenerativediseases, sarcopenia, muscle atrophy, inflammation, and cancer, or arelated disease or disorder thereof in a mammal, including a human, anda pharmaceutically acceptable excipient, carrier, or diluent.

In yet another aspect, the invention generally relates to a method fortreating, reducing, or preventing a disease or disorder. The methodincludes administering to a subject in need thereof a pharmaceuticalcomposition comprising an amount of an acid-base addition salt havingthe formula of:(B⁺)_(m)(Y⁻)_(n)  (II)wherein

(a) B⁺ is a cationic moiety of berberine or a derivative or analogthereof;

(b) Y⁻ is an anionic moiety of a pharmacologically active organic acid;and

(c) m and n are integers independently selected from 1, 2, 3, 4, 5 and 6so as to arrive at a charge neutral salt,

effective to treat, prevent, or reduce one or more diseases or disordersselected from metabolic disorders, heart diseases, neurodegenerativediseases, sarcopenia, muscle atrophy, inflammation, and cancer, or arelated disease or disorder thereof in a mammal, including a human, anda pharmaceutically acceptable excipient, carrier, or diluent.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Body weight of each treatment group at day 0 and day 14.

FIG. 2. Change of body weight of each treatment group after 14 days oftreatment.

FIG. 3. Blood glucose of each treatment group at day 2 and day 15.

FIG. 4. Change of blood glucose of each treatment group at day 2 and day15.

FIG. 5. ¹H NMR of metformin ursodeoxycholate in DMSO-D₆.

FIG. 6. IR spectrum of metformin ursodeoxycholate.

FIG. 7. ¹H NMR of berberine ursodeoxycholate (purified product).

FIG. 8. ¹H NMR of mixture of berberine hydrochloride (1.0 equiv.) andursodeoxycholic acid (1.0 equiv.) in DMSO-D₆.

FIG. 9. IR spectrum of berberine ursodeoxycholate (crude product).

The carbonyl stretching vibration band C═O of ursodeoxycholic acid atabout 1721 cm⁻¹ disappeared in IR spectrum of berberineursodeoxycholate.

FIG. 10. IR spectrum of mixture of berberine hydrochloride (1.0 equiv.)and ursodeoxycholic acid (1.0 equiv.). The carbonyl stretching vibrationband C═O of ursodeoxycholic acid appears at about 1719 cm⁻¹.

FIG. 11. Mass spectroscopy of berberine ursodeoxycholate: in negative MSmode, molecular mass of UDCA [M-H]⁻ 391.28 was identified.

FIG. 12. Mass spectroscopy of berberine ursodeoxycholate: in positive MSmode, molecular mass of BBR⁺ 336.14 was identified.

FIG. 13. (A) Plasma glucose concentrations during oral glucose tolerancetest (OGTT) and (B) the area under the OGTT glucose curve. Data areexpressed as the mean±S.E.M (n=7˜13). ** p<0.01 G2 vs. G1; # p<0.05 G4,G5, G6, or G7 vs. G2.

FIG. 14. Image of liver Sultan III staining in various groups (n=7-13).

FIG. 15. ¹H NMR of Berberine ursolic salt (400 MHz, DMSO-D6).

FIG. 16. The Effect of BUDCA on Serum LDL-c level, Serum HDL-c level,TC/HDL-c and AI of Hyperlipidemic Hamsters.

FIG. 17. The Effect of BUDCA on Serum AST Level of HyperlipidemicHamsters.

FIG. 18. The Effect of BUDCA on Serum ALT Level of HyperlipidemicHamsters.

FIG. 19. The Effect of BUDCA on Liver Weight and Liver Index ofHyperlipidemic Hamsters.

FIG. 20. The General Observation of Lipid Deposition in Liver Tissue.

FIG. 21. The Effect of BUDCA on TC and TG Content in Livers ofHyperlipidemic Hamsters.

FIG. 22. The Effect of BUDCA on Inflammation Score and Positive Area forOil Red 0.

DEFINITIONS

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. General principles of organicchemistry, as well as specific functional moieties and reactivity, aredescribed in “Organic Chemistry”, Thomas Sorrell, University ScienceBooks, Sausalito: 2006.

Certain compounds of the present invention may exist in particulargeometric or stereoisomeric forms. The present invention contemplatesall such compounds, including cis- and trans-isomers, R- andS-enantiomers, diastereomers, (D)-isomers, (L)-isomers, the racemicmixtures thereof, and other mixtures thereof, as falling within thescope of the invention. Additional asymmetric carbon atoms may bepresent in a substituent such as an alkyl group. All such isomers, aswell as mixtures thereof, are intended to be included in this invention.

Isomeric mixtures containing any of a variety of isomer ratios may beutilized in accordance with the present invention. For example, whereonly two isomers are combined, mixtures containing 50:50, 60:40, 70:30,80:20, 90:10, 95:5, 96:4, 97:3, 98:2, 99:1, or 100:0 isomer ratios arecontemplated by the present invention. Those of ordinary skill in theart will readily appreciate that analogous ratios are contemplated formore complex isomer mixtures.

If, for instance, a particular enantiomer of a compound of the presentinvention is desired, it may be prepared by asymmetric synthesis, or byderivation with a chiral auxiliary, where the resulting diastereomericmixture is separated and the auxiliary group cleaved to provide the puredesired enantiomers. Alternatively, where the molecule contains a basicfunctional group, such as amino, or an acidic functional group, such ascarboxyl, diastereomeric salts are formed with an appropriateoptically-active acid or base, followed by resolution of thediastereomers thus formed by fractional crystallization orchromatographic methods well known in the art, and subsequent recoveryof the pure enantiomers.

Given the benefit of this disclosure, one of ordinary skill in the artwill appreciate that synthetic methods, as described herein, may utilizea variety of protecting groups. By the term “protecting group”, as usedherein, it is meant that a particular functional moiety, e.g., O, S, orN, is temporarily blocked so that a reaction can be carried outselectively at another reactive site in a multifunctional compound. Inpreferred embodiments, a protecting group reacts selectively in goodyield to give a protected substrate that is stable to the projectedreactions; the protecting group should be selectively removable in goodyield by preferably readily available, non-toxic reagents that do notattack the other functional groups; the protecting group forms an easilyseparable derivative (more preferably without the generation of newstereogenic centers); and the protecting group has a minimum ofadditional functionality to avoid further sites of reaction. Oxygen,sulfur, nitrogen, and carbon protecting groups may be utilized. Examplesof a variety of protecting groups can be found in Protective Groups inOrganic Synthesis, Third Ed. Greene, T. W. and Wuts, P. G., Eds., JohnWiley & Sons, New York: 1999.

It will be appreciated that the compounds, as described herein, may besubstituted with any number of substituents or functional moieties.Throughout the specifications, groups and substituents thereof may bechosen to provide stable moieties and compounds.

As used herein, the term “effective amount” of an active agent refers toan amount sufficient to elicit the desired biological response. As willbe appreciated by those of ordinary skill in this art, the effectiveamount of a compound of the invention may vary depending on such factorsas the desired biological endpoint, the pharmacokinetics of thecompound, the disease being treated, the mode of administration, and thepatient.

As used herein, the term “treating, reducing, or preventing a disease ordisorder” refers to ameliorating such a condition before or after it hasoccurred. As compared with an equivalent untreated control, suchreduction or degree of prevention is at least 5%, 10%, 20%, 40%, 50%,60%, 80%, 90%, 95%, or 100% as measured by any standard technique.

As used herein, the term “pharmaceutically acceptable excipient,carrier, or diluent” refers to a pharmaceutically acceptable material,composition or vehicle, such as a liquid or solid filler, diluent,excipient, solvent or encapsulating material, involved in carrying ortransporting the subject pharmaceutical agent from one organ, or portionof the body, to another organ, or portion of the body. Each carrier mustbe “acceptable” in the sense of being compatible with the otheringredients of the formulation and not injurious to the patient. Someexamples of materials which can serve as pharmaceutically-acceptablecarriers include: sugars, such as lactose, glucose and sucrose;starches, such as corn starch and potato starch; cellulose, and itsderivatives, such as sodium carboxymethyl cellulose, ethyl cellulose andcellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients,such as cocoa butter and suppository waxes; oils, such as peanut oil,cottonseed oil, safflower oil, sesame oil, olive oil, corn oil andsoybean oil; glycols, such as propylene glycol; polyols, such asglycerin, sorbitol, mannitol and polyethylene glycol; esters, such asethyl oleate and ethyl laurate; agar; buffering agents, such asmagnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-freewater; isotonic saline; Ringer's solution; ethyl alcohol; phosphatebuffer solutions; and other non-toxic compatible substances employed inpharmaceutical formulations. Wetting agents, emulsifiers and lubricants,such as sodium lauryl sulfate, magnesium stearate, and polyethyleneoxide-polypropylene oxide copolymer as well as coloring agents, releaseagents, coating agents, sweetening, flavoring and perfuming agents,preservatives and antioxidants can also be present in the compositions.

As used herein, the terms “isolated” or “purified” refer to a materialthat is substantially or essentially free from components that normallyaccompany it in its native state. Purity and homogeneity are typicallydetermined using analytical chemistry techniques such as polyacrylamidegel electrophoresis or high performance liquid chromatography.

As used herein, the term “subject” refers to any animal (e.g., amammal), including, but not limited to humans, non-human primates,rodents, and the like, which is to be the recipient of a particulartreatment. Typically, the terms “subject” and “patient” are usedinterchangeably herein in reference to a human subject.

As used herein, the “an amount sufficient” refers to the amount of acompound, alone or in combination with another therapeutic regimen,required to treat, prevent, or reduce a metabolic disorder such asdiabetes in a clinically relevant manner. A sufficient amount of anactive compound used to practice the present invention for therapeutictreatment of conditions caused by or contributing to diabetes variesdepending upon the manner of administration, the age, body weight, andgeneral health of the mammal or patient. Ultimately, the prescriberswill decide the appropriate amount and dosage regimen. Additionally, aneffective amount may be an amount of compound in the combination of theinvention that is safe and efficacious in the treatment of a patienthaving a metabolic disorder such as diabetes over each agent alone asdetermined and approved by a regulatory authority (such as the U.S. Foodand Drug Administration).

As used herein, the “low dosage” refers to at least 5% less (e.g., atleast 10%, 20%, 50%, 80%, 90%, or even 95%) than the lowest standardrecommended dosage of a particular compound formulated for a given routeof administration for treatment of any human disease or condition. Forexample, a low dosage of an agent that reduces glucose levels and thatis formulated for administration by inhalation will differ from a lowdosage of the same agent formulated for oral administration.

As used herein, the “high dosage” is meant at least 5% (e.g., at least10%, 20%, 50%, 100%, 200%, or even 300%) more than the highest standardrecommended dosage of a particular compound for treatment of any humandisease or condition.

Isotopically-labeled compounds are also within the scope of the presentdisclosure. As used herein, an “isotopically-labeled compound” refers toa presently disclosed compound including pharmaceutical salts andprodrugs thereof, each as described herein, in which one or more atomsare replaced by an atom having an atomic mass or mass number differentfrom the atomic mass or mass number usually found in nature. Examples ofisotopes that can be incorporated into compounds presently disclosedinclude isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorous,fluorine and chlorine, such as ²H, ³H, ¹³C, ¹⁴C, ¹⁵N, ¹⁸O, ¹⁷O, ³¹P,³²P, ³⁵S, ¹⁸F, and ³⁶Cl, respectively.

By isotopically-labeling the presently disclosed compounds, thecompounds may be useful in drug and/or substrate tissue distributionassays. Tritiated (³H) and carbon-14 (¹⁴C) labeled compounds areparticularly preferred for their ease of preparation and detectability.Further, substitution with heavier isotopes such as deuterium (²H) canafford certain therapeutic advantages resulting from greater metabolicstability, for example increased in vivo half-life or reduced dosagerequirements and, hence, may be preferred in some circumstances.Isotopically labeled compounds presently disclosed, includingpharmaceutical salts, esters, and prodrugs thereof, can be prepared byany means known in the art.

Further, substitution of normally abundant hydrogen (¹H) with heavierisotopes such as deuterium can afford certain therapeutic advantages,e.g., resulting from improved absorption, distribution, metabolismand/or excretion (ADME) properties, creating drugs with improvedefficacy, safety, and/or tolerability. Benefits may also be obtainedfrom replacement of normally abundant ¹²C with ¹³C. See, WO 2007/005643,WO 2007/005644, WO 2007/016361, and WO 2007/016431.

Stereoisomers (e.g., cis and trans isomers) and all optical isomers of apresently disclosed compound (e.g., R and S enantiomers), as well asracemic, diastereomeric and other mixtures of such isomers are withinthe scope of the present disclosure.

Compounds of the present invention are, subsequent to their preparation,preferably isolated and purified to obtain a composition containing anamount by weight equal to or greater than 95% (“substantially pure”),which is then used or formulated as described herein. In certainembodiments, the compounds of the present invention are more than 99%pure.

Solvates and polymorphs of the compounds of the invention are alsocontemplated herein. Solvates of the compounds of the present inventioninclude, for example, hydrates.

Possible formulations include those suitable for oral, sublingual,buccal, parenteral (for example subcutaneous, intramuscular, orintravenous), rectal, topical including transdermal, intranasal andinhalation administration. Most suitable means of administration for aparticular patient will depend on the nature and severity of the diseaseor condition being treated or the nature of the therapy being used andon the nature of the active compound.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides various novel compositions of berberine incombination with pharmacologically active organic acids, and relatedmethods of their use in treating and/or preventing various diseases ordisorders. A noteworthy feature of the invention is the unique andsynergistic effect given rise by the combinations of berberine andselect pharmacologically active organic acids.

The invention also provides novel salts of ursodeoxycholic acid andorganic bases, pharmaceutical compositions thereof, as well as relatedmethods of preparation and use in treating and/or preventing variousliver diseases or disorders. Salts of ursodeoxycholic acid include thosewith organic bases such as berberine, metformin, carnitine, coptisine,palmatine, jatrorrhizine.

The invention further provides salts of berberine and organic acids,pharmaceutical compositions thereof, as well as related methods of theiruse in treating various diseases or disorders. Salts of berberineinclude those with organic acids such as R-(+)-α-lipoic acid,hydroxycitric acid, eicosapentaenoic acid, docosahexaenoic acid,docosapentaenoic acid, ursolic acid, corosolic acid, cinnamic acid,cholic acid, obeticholic acid, ursodeoxycholic acid, oleanolic acid,salicylic acid, betulinic acid, chlorogenic acid, caffeic acid, bassicacid, acetyl L-carnitine, S-allyl cysteine sulphoxide, S-methyl cysteinesulfoxide, pantothenic acid, ascorbic acid, retinoic acid, rhein,nicotinic acid, and biotin.

A central feature of the invention is the unique and synergistic effectgiven rise to by each of the two parts of the novel salts, i.e., apharmaceutically active cationic portion and a pharmaceutically activeanionic portion that collectively and synergistically target a diseaseor disorder with complementary mechanisms of actions and therebyproviding improved efficacies.

Diseases and disorders that may be treated and/or prevented by thecompounds, pharmaceutical compositions and methods disclosed hereininclude such as diabetes, diabetic complications, dyslipidemia,dyslipidemia in statin-intolerance patients, hyperlipidemia,hypercholesterolemia, hypertriglyceridemia, diabetic dyslipidemia,obesity, metabolic syndromes, pre-diabetes, atherosclerosis, heartdiseases, neurodegenerative diseases, sarcopenia, muscle atrophy,inflammation, cancer and liver diseases and conditions such as fattyliver, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis,cholestatic liver diseases or graft-versus-host disease of the liver.The compounds of this invention are also useful in improving liverfunctions in chronic viral associated liver diseases and alcohol-relatedliver diseases.

Combinations of Berberine or Derivative(s) and Pharmacologically ActiveOrganic Acids

The invention provides various novel compositions of berberine incombination with pharmacologically active organic acids, and relatedmethods of their use in treating various diseases or disorders. Theinvention thus embodies a unique approach that uses berberine insynergistic combinations with select pharmacologically active organicacids.

In one aspect, the invention generally relates to a compositioncomprising: (a) berberine or a derivative or analog thereof; (b) one ormore pharmacologically active organic acids; and (c) optionally apharmaceutically acceptable excipient, carrier, or diluent. Theberberine and the pharmacologically active organic acid(s) are presentin amounts that, when administered to a subject, are sufficient totreat, prevent, or reduce one or more diseases or disorders selectedfrom metabolic disorders, heart diseases, neurodegenerative diseases,muscle atrophy, liver diseases, inflammation, and cancer, or a relateddisease or disorder thereof in a mammal, including a human.

Berberine(5,6-dihydro-9,10-dimethoxybenzo[g]-1,3-benzodioxolo[5,6-a]quinolizinium),an isoquinoline alkaloid isolated from Rhizoma Coptidis, has had a longhistory of medicinal use in China to treat various gastrointestinaldiseases. Berberine is found in a variety of plants as Berberis,Hydrastis canadensis, Xanthorhiza simplicissima, Phellodendron amurense,Coptis chinensis, Tinospora cordifolia, Argemone mexicana, andEschscholzia californica. In the past two decades, in vitro and in vivostudies have demonstrated the efficacy of berberine when used alone oras a combination for diabetes, dyslipidemia, cancer, neuroprotection andcardiovascular diseases. Currently, berberine can be obtainedcommercially in the form of chloride, sulfate or tannate salt, withberberine hydrochloride having been used in almost all previous studies.The low bioavailability of berberine in the current available formsmakes its applications for the treatment of chronic and systemic diseasevery challenging.

R-(+)-α-Lipoic acid ((R)-6,8-Dithiooctanoic acid, (R)-6,8-Thioctic acid,(R)-(+)-1,2-Dithiolane-3-pentanoic acid) was identified as a catalyticagent for oxidative decarboxylation of pyruvate and α-ketoglutarate. Inhuman, R-(+)-α-lipoic acid exists in the body as a portion of severalmulti-enzyme complexes involved in energy formation and is an essentialcomponent of mitochondrial respiratory enzymes. R-(+)-α-Lipoic acid isbest known for its potent anti-oxidant effects and has been used for thetreatment of diabetic neuropathy, degenerative neuronal disease,atherosclerosis and other abnormalities related to oxidative stress.

Hydroxycitric acid (1,2-dihydroxypropane-1,2,3-tricarboxylic acid) is aderivative of citric acid found in a variety of tropical plantsincluding Garcinia cambogia and Hibiscus subdariffa. Hydroxycitric acidis the active component of Garcinia cambogia extract, which has beenwidely utilized as dietary supplement for weight loss. There have beenreports on hydroxycitric acid's effects in improving glucose tolerance,providing liver protection against toxicity associated with ethanol anddexamethasone, and controlling blood pressure. In addition, the compoundhas been found to reduce markers of inflammation in brain, intestines,kidney and serum.

Eicosapentaenoic acid (EPA or(5Z,8Z,11Z,14Z,17Z)-5,8,11,14,17-icosapentaenoic acid), anddocosahexaenoic acid (DHA,4Z,7Z,10Z,13Z,16Z,19Z)-docosa-4,7,10,13,16,19-hexaenoic acid), are twobest-investigated omega-3 polyunsaturated fatty acids. EPA is the activemolecule in two FDA-approved anti-hypertriglyceridemic agents. It hasbeen demonstrated that EPA and DHA can reduce free fatty acid andtriglyceride synthesis and increase their disposal. Effects of EPA andDHA have also been demonstrated in reducing chronic inflammation,improving insulin resistance, maintaining heart and vascular health andreducing the risk of coronary heart disease. In addition to EPA and DHA,many more omega-3 fatty acids existed in nature with a range oftherapeutic benefits, include but not limited to Docosapentaenoic acid(DPA), α-Linolenic acid (ALA), Eicosatrienoic acid (ETE) etc.

Ursolic acid((1S,2R,4aS,6aR,6aS,6bR,8aR,10S,12aR,14bS)-10-hydroxy-1,2,6a,6b,9,9,12a-heptamethyl-2,3,4,5,6,6a,7,8,8a,10,11,12,13,14b-tetradecahydro-1H-picene-4a-carboxylicacid) and corosolic acid((1S,2R,4aS,6aR,6aS,6bR,8aR,10R,11R,12aR,14bS)-10,11-Dihydroxy-1,2,6a,6b,9,9,12a-heptamethyl-2,3,4,5,6,6a,7,8,8a,10,11,12,13,14b-tetradecahydro-1H-picene-4a-carboxylicacid) are members of the pentacyclic triterpene acid class of compoundswidely distributed in the plant kingdom. They have been shown to exhibitfavorable pharmacological effects both in vivo and in vitro, includingglucose reduction, anti-obesity, anti-inflammatory, reduce muscleatrophy, anti-cancer, liver protection, anti-oxidative stress.

Cinnamic acid, cholic acid, obeticholic acid, ursodeoxycholic acid,oleanolic acid, salicylic acid, betulinic acid, chlorogenic acid,caffeic acid, bassic acid, acetyl L-carnitine, S-allyl cysteinesulphoxide, S-methyl cysteine sulfoxide, pantothenic acid, ascorbicacid, retinoic acid, rhein, nicotinic acid and biotin in either apurified form or an active extract (Table 1) are additional organicacids with demonstrated pharmacologically activities in the treatment orprevention of diabetes, diabetic complications, dyslipidemia, obesity,metabolic syndromes, pre-diabetes, heart diseases, fatty liver, NAFLD,NASH, muscle atrophy, inflammation, and cancers.

Exemplary pharmacologically active organic acids are listed in Table 1.

TABLE 1 Exemplary Pharmacologically Active Organic Acids Name IUPAC NameStructure Cinnamic acid (E)-3-phenylprop-2-enoic acid

Cholic acid (R)-4- ((3R,5S,7R,8R,9S,10S,12S, 13R,14S,17R)-3,7,12-trihydroxy-10,13- dimethylhexadecahydro-1H-cyclopenta[a]phenanthren- 17-yl)pentanoic acid

Obeticholic acid (3α,5β,6α,7α)-6-Ethyl-3,7- dihydroxycholan-24-oic acid

Ursodeoxycholic acid 3α,7β-dihydroxy-5β-cholan- 24-oic acid OR (R)-4-((3R,5S,7S,8R,9S,10S,13R, 14S,17R)-3,7-dihydroxy- 10,13-dimethylhexadecahydro- 1H- cyclopenta[a]phenanthren- 17-yl)pentanoicacid

Oleanolic acid (4aS,6aR,6aS,6bR,8aR,10S, 12aR,14bS)-10-hydroxy-2,2,6a,6b,9,9,12a- heptamethyl- 1,3,4,5,6,6a,7,8,8a,l 0,11,12, 13,14b-tetradecahydropicene-4a- carboxylic acid

Salicylic acid 2-Hydroxybenzoic acid

Betulinic acid (3β)-3-Hydroxy-lup-20(29)- en-28-oic acid

Chlorogenic acid (1S,3R,4R,5R)-3-{[(2Z)-3- (3,4-dihydroxyphenyl)prop-2-enoyl]oxy}-1,4,5- trihydroxycyclohexane- carboxylic acid

Caffeic acid 3-(3,4-Dihydroxyphenyl)-2- propenoic acid 3,4-Dihydroxy-cinnamic acid trans-Caffeate 3,4- Dihydroxy-trans- cinnamate)(E)-3-(3,4- dihydroxyphenyl)-2-

propenoic acid 3,4- Dihydroxybenzeneacrylic acid3-(3,4-Dihydroxyphenyl)- 2-propenoic acid Bassic acid(4aR,6bS,9R,10R,11S,12aR, 14bS)-10,11-dihydroxy-9- (hydroxymethyl)-2,2,6b,9,12a-pentamethyl- 1,2,3,4,4a,5,6,6a,6b,7,9,10,11,12,12a,12b,13,14b- octadecahydropicene-4a- carboxylic acid

Acetyl L- carnitine (R)-3-Acetyloxy-4- trimethylammonio- butanoate

S-allyl-L- cysteine sulphoxide (2R)-2-amino-3-[(S)-prop-2-enylsulfinyl]propanoic acid

S-methyl-L- cysteine sulfoxide 3-(methylsulfinyl)-L-alanine

Pantothenic acid 3-[(2,4-Dihydroxy-3,3- dimethylbutanoyl)amino]propanoic acid

Ascorbic acid (5R)-[(1S)-1,2- dihydroxyethyl]-3,4-dihydroxyfuran-2(5H)-one

Retinoic acid (2E,4E,6E,8E)-3,7- dimethyl-9-(2,6,6-trimethylcyclohexen-1- yl)nona-2,4,6,8-tetraenoic acid

Rhein 4,5-dihydroxy-9,10- dioxoanthracene-2- carboxylic acid

Nicotinic acid pyridine-3-carboxylic acid

Biotin 5-[(3aS,4S,6aR)-2- oxohexahydro-1H- thieno[3,4-d]imidazol-4-yl]pentanoic acid

Exemplary berberine derivatives or analogs are listed in Table 2.

TABLE 2 Berberine Derivatives or Analogs

R₁ = R₂ = R₃ = R₄ = CH₃

R = H

R = C₈-C₁₂ alkyl

R₁ = OH, carbonyl; R₂, R₃ = H, carbonyl; n = 2-6; X = O R₁ = OH,carbonyl; R₂, R₃ = H, OH, carbonyl; n = 2-6; X = NH

R₁, R₃, R₂, R₄ = OH, C₁-C₆ alkoxy, OCH₂O G = Z—Ar, Y—Ar₂ Z = O(CH₂)_(m),CONH(CH₂)_(m), NHCO(CH₂)_(m) Y = O(CH₂)_(m)CH, CONH(CH₂)_(m)CH,NHCO(CH₂)_(m)CH n = 1-5; m = 1-3; Ar = 5-15 membered unsaturated oraromatic ring

R₁, R₃, R₂, R₄ = OH, C₁-C₆ alkoxy, OCH₂O G = Z—Ar, Y—Ar₂ Z = O(CH₂)_(m),CONH(CH₂)_(m), NHCO(CH₂)_(m) Y = O(CH₂)_(m)CH, CONH(CH₂)_(m)CH,NHCO(CH₂)_(m)CH n = 1-5; m = 1-3; Ar = 5-15 membered unsaturated oraromatic ring

R₁, R₃, R₂, R₄ = OH, C₁-C₆ alkoxy, OCH₂O G = Z—Ar, Y—Ar₂ Z = O(CH₂)_(m),CONH(CH₂)_(m), NHCO(CH₂)_(m) Y = O(CH₂)_(m)CH, CONH(CH₂)_(m)CH,NHCO(CH₂)_(m)CH n = 1-5; m = 1-3; Ar = 5-15 membered unsaturated oraromatic ring

R₁, R₃, R₂, R₄ = OH, C₁-C₆ alkoxy, OCH₂O G = Z—Ar, Y—Ar₂ Z = O(CH₂)_(m),CONH(CH₂)_(m), NHCO(CH₂)_(m) Y = O(CH₂)_(m)CH, CONH(CH₂)_(m)CH,NHCO(CH₂)_(m)CH n = 1-5; m = 1-3; Ar = 5-15 membered unsaturated oraromatic ring

R₁, R₃, R₂, R₄ = OH, C₁-C₆ alkoxy, OCH₂O G = Z—Ar, Y—Ar₂ Z = O(CH₂)_(m),CONH(CH₂)_(m), NHCO(CH₂)_(m) Y = O(CH₂)_(m)CH, CONH(CH₂)_(m)CH,NHCO(CH₂)_(m)CH n = 1-5; m = 1-3; Ar = 5-15 membered unsaturated oraromatic ring

R₁, R₂, R₃, R₄ = OCH₃, OH, OCH₂O

R₁ = H, Me R₂ = Bn, 3,5-dinitrobenzyl

X = F, Cl, Br, I, SO₄, NO₃, PO₄, citrate, acetate, lactate R₁ and R₂ =independently alkyl; R₃ = H, F, Cl, Br, or I

X = F, Cl, Br, I, SO₄, NO₃, PO₄, citrate, acetate, lactate R₁ and R₂ =independently alkyl; R₃ = H, F, Cl, Br, or I

Y = CH₂, —C═O, —C═S; X = C having a linear, branched,saturated/unsaturated linear structure; n = 1-10

Y = CH₂, —C═O, —C═S; X = C having a linear, branched,saturated/unsaturated linear structure; n = 1-10

Y = CH₂, —C═O, —C═S; X = C having a linear, branched,saturated/unsaturated linear structure; n = 1-10

R = glucosyl, mannosyl, maltosyl, lactosyl, galactosyl, fructosyl,xylosyl, arabinosyl X = Cl, Br, I

R₁, R₂ = H, C₁-C₄ alkoxy, OCH₂O R₃ = C₁-C₈ alkyl R₄, R₅ = C₁-C₂ alkoxy

R₁, R₂ = H, C₁-C₄ alkoxy, OCH₂O R₃ = CN, COOR₆ (R₆ = C₁-C₂ alkyl) R₄, R₅= C₁-C₂ alkoxy

R₁, R₂ = H, C₁-C₄ alkoxy, OCH2O R₃ = C₁-C₂ alkyl, phenyl R₄, R₅ = C₁-C₂alkoxy

R₁, R₂ = H, (CH₂)_(n-6)CO₂R′, C(O)R″, OR′, NR₁₀R₁₁, C(O)NR₁₀R₁₁, alkylR₁R₂ = OCH₂CH₂O; R₃, R₈ = H, OH, Cl, Br, F, I, CN, NH₂, (O)NH₂, CO₂H,alkyl; R₃′ = H; R₃R₃′= O, R₄ = H, halogen, OR′, OSO₂R″, OC(O)R″, OCO₂R″OC(O)NR′R″, O-alkylene-NR′R″, O-alkylene-OSO₂R″, O-alkylene-NR′SO₂R″,O-alkylene-NR′COR′, alkyl; R₅, R₆ = H, halogen, OH, alkoxy R₄R₅ = OCH₂O;R₅R₆ = OCH₂O; R₇ = H, OH, halogen, alkyl or alkoxy R₁₀, R₁₁ = H, CO₂R″,alkyl

R = SO₂C₆H₄-3-F

X = (CH₂)_(m), (CH₂)_(m)CO; n = 2-10; m = 1-9 Y = NR₁Ar, OAr; Ar =substituted aryl R₁ = H, Me, Et, Pr, i-Pr; Z = F, Cl, Br, I

R = 2-acetic acid Me ester, 3-acetic Me ester, 4-acetic Me ester,2-acetic Me Et ester, 3-acetic Me Et ester, 4-acetic Me Et ester,2-acetate, 3-acetate, 4-acetate, 2-acetate potassium, 3-acetatepotassium, 4-acetate potassium; n = 2-6

In certain embodiments, the pharmacologically active organic acid(s) isone or more agents selected from the group consisting of R-(+)-α-lipoicacid, hydroxycitric acid, eicosapentaenoic acid, docosahexaenoic acid,docosapentaenoic acid, ursolic acid, corosolic acid, cinnamic acid,cholic acid, obeticholic acid, ursodeoxycholic acid, oleanolic acid,salicylic acid, betulinic acid, chlorogenic acid, caffeic acid, bassicacid, acetyl L-carnitine, S-allyl cysteine sulphoxide, S-methyl cysteinesulfoxide, pantothenic acid, ascorbic acid, retinoic acid, rhein,nicotinic acid, biotin and other organic acid that is generallyrecognized pharmacologically active for one or more diseases ordisorders selected from metabolic disorders, heart diseases,neurodegenerative diseases, liver diseases, muscle atrophy,inflammation, and cancer, or a related disease or disorder thereof in amammal, including a human by those of skill in the art.

In certain embodiments, the composition further includes one or moreadditional agent(s) selected from the group consisting of vitamin D,vitamin C, vitamin E, vitamin B12, vitamin A, benfotiamine, chromiumpicolinate and vanadium.

In certain embodiments, the disease or disorder is a metabolic disorderand is selected from diabetes, diabetic complications, dyslipidemia,obesity, metabolic syndromes, pre-diabetes, fatty liver, NAFLD and NASH.In certain embodiments, the disease or disorder is heart diseases. Incertain embodiments, the disease or disorder is neurodegenerativediseases. In certain embodiments, the disease or disorder is cancer. Incertain embodiments, the cancer is selected from the group consisting ofbreast cancer, prostate cancer, lung cancer, hepatocellular carcinoma,pancreatic cancer, gastric carcinoma, colorectal cancer, leukemia,multiple myeloma, melanoma and glioblastoma. In certain embodiments, thedisease or disorder is muscle atrophy. In certain embodiments, thedisease or disorder is muscle atrophy is selected from skeletal muscleatrophy.

In certain embodiments, the composition further includes apharmaceutically acceptable excipient, carrier, or diluent.

In certain preferred embodiments, the composition includes berberine andR-(+)-α-Lipoic acid. In certain preferred embodiments, the compositionincludes berberine, R-(+)-α-Lipoic acid and vitamin D. In certainpreferred embodiments, the composition includes berberine,R-(+)-α-Lipoic acid and vitamin B12. In certain preferred embodiments,the composition includes berberine, R-(+)-α-Lipoic acid, vitamin B12 andbenfotiamine. In certain preferred embodiments, the composition includesberberine, R-(+)-α-Lipoic acid, vitamin B12, benfotiamine and omega-3polyunsaturated fatty acids.

In certain preferred embodiments, the composition includes berberine andhydroxycitric acid. In certain preferred embodiments, the compositionincludes berberine, hydroxycitric acid and vitamin D. In certainpreferred embodiments, the composition includes berberine, hydroxycitricacid, vitamin D and omega-3 polyunsaturated fatty acids. In certainpreferred embodiments, the composition includes berberine, extracts fromGarcinia cambogia or Hibiscus subdariffa (hydroxycitric acid), vitamin Dand omega-3 polyunsaturated fatty acids.

In certain preferred embodiments, the composition includes berberine andone or both of EPA and DHA. In certain preferred embodiments, thecomposition includes berberine, one or both of EPA and DHA and vitaminD.

In certain preferred embodiments, the composition includes berberine andursolic acid. In certain preferred embodiments, the composition includesberberine, ursolic acid and/or corosolic acid and vitamin D. In certainpreferred embodiments, the composition includes berberine, ursolic acidand/or corosolic acid, vitamin D and omega-3 polyunsaturated fattyacids. In certain preferred embodiments, the composition includesberberine, banaba extracts (corosolic acid), Holy Basil or apple peelsextracts (ursolic acid), vitamin D and omega-3 polyunsaturated fattyacids.

In certain preferred embodiments, the composition includes berberine andcinnamic acid. In certain preferred embodiments, the compositionincludes berberine, cinnamic acid and vitamin D. In certain preferredembodiments, the composition includes berberine, cinnamic acid, vitaminD and omega-3 polyunsaturated fatty acids.

In certain preferred embodiments, the composition includes berberine andbile acid(s) (e.g., cholic acid, obeticholic acid and/or ursodeoxycholicacid). In certain preferred embodiments, the composition includesberberine, bile acid(s) (e.g., cholic acid, obeticholic acid and/orursodeoxycholic acid) and vitamin D. In certain preferred embodiments,the composition includes berberine, bile acid(s) (e.g., cholic acid,obeticholic acid and/or ursodeoxycholic acid), vitamin D and omega-3polyunsaturated fatty acids.

In certain preferred embodiments, the composition includes berberine andoleanolic acid. In certain preferred embodiments, the compositionincludes berberine, oleanolic acid and vitamin D. In certain preferredembodiments, the composition includes berberine, oleanolic acid, vitaminD and omega-3 polyunsaturated fatty acids.

In certain preferred embodiments, the composition includes berberine andsalicylic acid. In certain preferred embodiments, the compositionincludes berberine, salicylic acid and vitamin D. In certain preferredembodiments, the composition includes berberine, salicylic acid, vitaminD and omega-3 polyunsaturated fatty acids.

In certain preferred embodiments, the composition includes berberine andbetulinic acid. In certain preferred embodiments, the compositionincludes berberine, betulinic acid and vitamin D. In certain preferredembodiments, the composition includes berberine, betulinic acid, vitaminD and omega-3 polyunsaturated fatty acids.

In certain preferred embodiments, the composition includes berberine andchlorogenic acid. In certain preferred embodiments, the compositionincludes berberine, chlorogenic acid and vitamin D. In certain preferredembodiments, the composition includes berberine, chlorogenic acid,vitamin D and omega-3 polyunsaturated fatty acids.

In certain preferred embodiments, the composition includes berberine andcaffeic acid. In certain preferred embodiments, the composition includesberberine, caffeic acid and vitamin D. In certain preferred embodiments,the composition includes berberine, caffeic acid, vitamin D and omega-3polyunsaturated fatty acids.

In certain preferred embodiments, the composition includes berberine andbassic acid. In certain preferred embodiments, the composition includesberberine, bassic acid and vitamin D. In certain preferred embodiments,the composition includes berberine, bassic acid, vitamin D and omega-3polyunsaturated fatty acids.

In certain preferred embodiments, the composition includes berberine andacetyl L-carnitine. In certain preferred embodiments, the compositionincludes berberine, acetyl L-carnitine and vitamin D. In certainpreferred embodiments, the composition includes berberine, acetylL-carnitine, vitamin D and omega-3 polyunsaturated fatty acids.

In certain preferred embodiments, the composition includes berberine andS-allyl cysteine sulphoxide and/or S-methyl cysteine sulfoxide. Incertain preferred embodiments, the composition includes berberine,5-allyl cysteine sulphoxide and/or S-methyl cysteine sulfoxide andvitamin D. In certain preferred embodiments, the composition includesberberine, S-allyl cysteine sulphoxide and/or S-methyl cysteinesulfoxide, vitamin D and omega-3 polyunsaturated fatty acids.

In certain preferred embodiments, the composition includes berberine andpantothenic acid. In certain preferred embodiments, the compositionincludes berberine, pantothenic acid and vitamin D. In certain preferredembodiments, the composition includes berberine, pantothenic acid,vitamin D and omega-3 polyunsaturated fatty acids.

In certain preferred embodiments, the composition includes berberine andascorbic acid. In certain preferred embodiments, the compositionincludes berberine, ascorbic acid and vitamin D. In certain preferredembodiments, the composition includes berberine, ascorbic acid, vitaminD and omega-3 polyunsaturated fatty acids.

In certain preferred embodiments, the composition includes berberine andretinoic acid. In certain preferred embodiments, the compositionincludes berberine, retinoic acid and vitamin D. In certain preferredembodiments, the composition includes berberine, retinoic acid, vitaminD and omega-3 polyunsaturated fatty acids.

In certain preferred embodiments, the composition includes berberine andrhein. In certain preferred embodiments, the composition includesberberine, rhein and vitamin D. In certain preferred embodiments, thecomposition includes berberine, rhein, vitamin D and omega-3polyunsaturated fatty acids. In certain preferred embodiments, thecomposition includes berberine, rhubarb extracts (rhein), vitamin D andomega-3 polyunsaturated fatty acids.

In certain preferred embodiments, the composition includes berberine andnicotinic acid. In certain preferred embodiments, the compositionincludes berberine, nicotinic acid and vitamin D. In certain preferredembodiments, the composition includes berberine, nicotinic acid, vitaminD and omega-3 polyunsaturated fatty acids.

In certain preferred embodiments, the composition includes berberine andbiotin. In certain preferred embodiments, the composition includesberberine, biotin and vitamin D. In certain preferred embodiments, thecomposition includes berberine, biotin, vitamin D and omega-3polyunsaturated fatty acids.

In another aspect, the invention generally relates to a method fortreating, reducing, or preventing a metabolic disorder. The methodincludes administering to a subject in need thereof a pharmaceuticalcomposition, which includes: (a) berberine or a derivative or analogthereof; (b) one or more pharmacologically active organic acids, in atherapeutically effective amount, and (c) optionally a pharmaceuticallyacceptable excipient, carrier, or diluent.

In certain embodiments, the metabolic disorder is selected fromdiabetes, diabetic complications, dyslipidemia, diabetic dyslipidemia,dyslipidemia in statin-intolerance patients, hyperlipidemia, obesity,metabolic syndromes, pre-diabetes, fatty liver, NAFLD and NASH.

In certain preferred embodiments, the metabolic disorder is type 2diabetes.

In certain preferred embodiments, the diabetic complications arediabetic neuropathy, diabetic retinopathy or diabetic nephropathy.

In certain preferred embodiments, the hyperlipidemia ishypercholesterolemia, hypertriglyceridemia, or both.

In certain preferred embodiments, the pharmacologically active organicacid is selected from the group consisting of R-(+)-α-lipoic acid,hydroxycitric acid, eicosapentaenoic acid, docosahexaenoic acid,docosapentaenoic acid, ursolic acid, corosolic acid, cinnamic acid,cholic acid, obeticholic acid, ursodeoxycholic acid, oleanolic acid,salicylic acid, betulinic acid, chlorogenic acid, caffeic acid, bassicacid, acetyl L-carnitine, S-allyl cysteine sulphoxide, S-methyl cysteinesulfoxide, pantothenic acid, ascorbic acid, retinoic acid, rhein,nicotinic acid and biotin. In certain embodiments, the pharmaceuticalcomposition further comprises a third agent selected from the groupconsisting of vitamin D, vitamin C, vitamin E, vitamin B12, vitamin A,benfotiamine, chromium picolinate and vanadium.

In certain preferred embodiments of the method, the subject suffers fromdiabetes and diabetic complications and the pharmaceutical compositioncomprises berberine and R-(+)-α-lipoic acid. In certain preferredembodiments of the method, the subject suffers from diabetic nephropathyand the pharmaceutical composition comprises berberine and rhein (orrhubarb extracts). In certain preferred embodiments of the method, thesubject suffers from diabetes and obesity and the pharmaceuticalcomposition comprises berberine and hydroxycitric acid (or GarciniaCambogia extracts). In certain preferred embodiments of the method, thesubject suffers from diabetes and dyslipidemia and the pharmaceuticalcomposition comprises berberine and one or more of EPA, DHA and DPA.

In certain preferred embodiments of the method, the subject suffers fromdiabetes and muscle atrophy and the pharmaceutical composition comprisesberberine and one or both of ursolic acid and corosolic acid. In certainpreferred embodiments of the method, the subject suffers from diabetesand muscle atrophy, and the pharmaceutical composition comprisesberberine and one or both of Holy Basil or apple peels extracts (ursolicacid) and banaba extracts (corosolic acid).

In certain preferred embodiments of the method, the subject suffers fromfatty liver, NAFLD and NSAH, and the pharmaceutical compositioncomprises berberine and one or more of cholic acid, obeticholic acid andursodeoxycholic acid. In certain preferred embodiments of the method,the subject suffers from fatty liver, NAFLD and NSAH, and thepharmaceutical composition comprises berberine and bile acids.

In certain preferred embodiments of the method, the pharmaceuticalcomposition includes further comprises vitamin D.

In certain preferred embodiments of the method, the pharmaceuticalcomposition further includes vitamin E.

In certain preferred embodiments of the method, the pharmaceuticalcomposition further includes vitamin B12.

In certain preferred embodiments of the method, the pharmaceuticalcomposition further includes benfotiamine.

In certain preferred embodiments of the method, the pharmaceuticalcomposition further includes vitamin C.

In certain preferred embodiments of the method, the pharmaceuticalcomposition further includes vitamin A.

In certain preferred embodiments of the method, the pharmaceuticalcomposition further includes benfotiamine.

In certain preferred embodiments of the method, the pharmaceuticalcomposition further includes chromium picolinate.

In certain preferred embodiments of the method, the pharmaceuticalcomposition further includes vanadium.

In certain preferred embodiments of the method, treating, reducing, orpreventing a metabolic disorder is by reducing blood glucose levels ofthe subject. In certain preferred embodiments of the method, treating,reducing, or preventing a metabolic disorder is by reducing totalcholesterol (TC), triglyceride (TG) and low-density lipoproteincholesterol (LDL-c) levels, increasing high-density lipoproteincholesterol (HDL-c) levels of the subject. In certain preferredembodiments of the method, treating, reducing, or preventing a metabolicdisorder is by normalizing liver enzyme levels of the subject. Incertain preferred embodiments of the method, treating, reducing, orpreventing a metabolic disorder is by altering insulin signaling pathwaysuch that glucose levels are reduced. In certain preferred embodimentsof the method, treating, reducing, or preventing a metabolic disorder isby regulating multiple metabolic pathways such as increasing secretionof insulin, improving insulin sensitivity, reducing gluconeogenesis inliver, reducing glucose absorption, ameliorating dyslipidemia,anti-inflammation to achieve the desired pharmacological effects.

In yet another aspect, the invention generally relates to a kit thatincludes: (i) an agent of berberine or a derivative or analog thereof;(ii) one or more agent(s) selected from pharmaceutically active organicacids; and (iii) instructions for administering the combined agents to apatient having or at risk of having one or more diseases or disordersselected from metabolic disorders, heart diseases, neurodegenerativediseases, liver diseases, muscle atrophy, and cancer.

In certain embodiments, the derivative or analog of berberine isselected Table 2. In certain embodiments, additional agent is selectedfrom any one or more of the agents of R-(+)-α-lipoic acid, hydroxycitricacid, eicosapentaenoic acid, docosahexaenoic acid, docosapentaenoicacid, ursolic acid, corosolic acid, cholic, ursodeoxycholic acid or theothers listed in Table 1.

Salts of Ursodeoxycholic Acid or Derivatives

The invention also provides novel salts of ursodeoxycholic acid andorganic bases, pharmaceutical compositions thereof, as well as relatedmethods of preparation and use in treating and/or preventing variousliver diseases or disorders, and metabolic disorders. Salts ofursodeoxycholic acid include those with organic bases such as berberine,metformin, carnitine, coptisine, palmatine, jatrorrhizine.

In yet another aspect, the present invention generally relates to anacid-base addition salt in substantially pure form, having the formulaof:(X⁺)_(m)(U⁻)_(n)  (I)wherein

(a) U⁻ is an anionic moiety of ursodeoxycholic acid or a derivative oranalog thereof;

(b) X⁺ is a cationic moiety of a pharmacologically active organic base;and

(c) m and n are integers independently selected from 1, 2, 3, 4, 5 and 6so as to arrive at a charge neutral salt.

U⁻ can be an anionic moiety of any suitable derivative or analog ofursodeoxycholic, for example, selected from Table 3.

X⁺ can be a cationic moiety of any suitable pharmacologically activeorganic base. In certain embodiments, for example, the pharmacologicallyactive organic base may be selected from the group consisting ofberberine, metformin, carnitine and coptisine, palmatine, jatrorrhizine.In certain embodiments, X⁺ can also be a cationic moiety of otherorganic base that is generally recognized pharmacologically active forone or more diseases or disorders selected from various liver diseasesor disorders such as fatty liver, NAFLD, NASH, cholestatic liverdiseases, graft-versus-host disease of the liver, chronic viralassociated liver diseases, alcohol-related liver diseases, metabolicdiseases or disorders such as pre-diabetes, diabetes, dyslipidemia,diabetic dyslipidemia, hyperlipidemia, obesity, or a related disease ordisorder thereof in a mammal, including a human.

Ursodeoxycholic acid (UDCA or ursodiol, with the chemical names of3α,7β-dihydroxy-5β-cholan-24-oic acid or(R)-4-((3R,5S,7S,8R,9S,10S,13R,14S,17R)-3,7-dihydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)pentanoicacid) is a secondary bile acid, a substance naturally produced by thebody that is stored in the gallbladder. Ursodiol is used to dissolvegallstones in patients as an alternative to surgery. Ursodiol is alsoused to prevent the formation of gallstones in overweight patients whoare losing weight very quickly. Ursodiol works by decreasing theproduction of cholesterol and by dissolving the cholesterol in bile sothat it cannot form stones. Ursodiol is also the first-line therapy forthe treatment of PBC, PSC and cholestatic liver diseases. There havebeen limited studies of ursodiol on NASH, but the results werecontradictory and inconclusive. Thus, the effect of ursodiol on NASHremains unclear.

Metformin (N,N-Dimethylimidodicarbonimidic diamide) is a potentanti-hyperglycemic agent now recommended as the first line oral therapyfor type 2 diabetes (T2D). The main effect of this drug is to acutelydecrease hepatic glucose production, mostly through a mild and transientinhibition of the mitochondrial respiratory-chain complex 1. Inaddition, the resulting decrease in hepatic energy status activates theAMP-activated protein kinase (AMPK), a cellular metabolic sensor,providing a generally accepted mechanism for metformin action on hepaticgluconeogenic program. Beyond its effect on glucose metabolism,metformin was reported to restore ovarian function in polycystic ovarysyndrome, to reduce fatty liver and to lower microvascular andmacrovascular complications associated with T2D. Its use was alsorecently suggested as an adjuvant treatment for cancer or gestationaldiabetes, and for the prevention in pre-diabetic populations. Studies ofmetformin for NAFLD and NASH have multiplied in the past few years,however, its efficacy for NAFLD and NASH remains to be approved.

Coptisine [6,7-Dihydro-bis(1,3)benzodioxolo(5,6-a:4′,5′-g)quinolizinium], palmatine[2,3,9,10-tetramethoxy-5,6-dihydroisoquinolino[2,1-b]isoquinolin-7-ium],and jatrorrhizine[2,9,10-trimethoxy-5,6-dihydroisoquinolino[2,1-b]isoquinolin-7-ium-3-ol]are naturally alkaloids that have demonstrated similar pharmacologicalproperties as berberine in previous studies.

L-Carnitine is a naturally occurring amino acid. It is biosynthesized inthe liver and kidneys from lysine and methionine. L-Carnitine plays animportant role in the metabolism of fat, functioning as a transporter offatty acids into the mitochondria.

Exemplary derivatives or analogs of ursodeoxycholic acid are listed inTable 3.

TABLE 3 Ursodeoxycholic acid Derivatives or Analogs

R₁ is selected from the group consisting of C₁-C₄ alkyl or a halogen; oran ester

R1 = αOH, R2 = OH R1 = βOH, R2 = H R1 = αOH, R2 = H R1 = H, R2 = OH R1 =H, R2 = H

a: 7α b: 7β

V

(I)

R′ R is a radical selected from —CH2— SO₃H and —COOH and R′ is a radicalselected from —H and —(CH₂)₂—CONH, —CH₂— CONH₁, —(CH₂)₂—SCH₃, —CH₂—S—CH₂—COOH, respectively

In certain preferred embodiments, U⁻ is an anionic moiety ofursodeoxycholic acid, X⁺ is a cationic moiety of berberine or aderivative or analog thereof, and m=1 and n=1. Exemplary derivatives oranalogs of berberine are listed in Table 2. In certain preferredembodiments, U⁻ is an anionic moiety of ursodeoxycholic acid, X⁺ is acationic moiety of berberine, and m=1 and n=1.

In certain preferred embodiments, U⁻ is an anionic moiety ofursodeoxycholic acid, X⁺ is a cationic moiety of metformin or aderivative or analog thereof, and m=1 and n=1. Exemplary derivatives oranalogs of metformin are listed in Table 4. In certain preferredembodiments, U⁻ is an anionic moiety of ursodeoxycholic acid, X⁺ is acationic moiety of metformin, and m=1 and n=1.

In certain preferred embodiments, U⁻ is an anionic moiety ofursodeoxycholic acid, X⁺ is a cationic moiety of carnitine or aderivative or analog thereof, and m=1 and n=1. Exemplary derivatives oranalogs of carnitine are listed in Table 5. In certain preferredembodiments, U⁻ is an anionic moiety of ursodeoxycholic acid, X⁺ is acationic moiety of carnitine, and m=1 and n=1.

In certain preferred embodiments, U⁻ is an anionic moiety ofursodeoxycholic acid, X⁺ is a cationic moiety of coptisine or aderivative or analog thereof, and m=1 and n=1.

In certain preferred embodiments, U⁻ is an anionic moiety ofursodeoxycholic acid, X⁺ is a cationic moiety of palmatine or aderivative or analog thereof, and m=1 and n=1.

In certain preferred embodiments, U⁻ is an anionic moiety ofursodeoxycholic acid, X⁺ is a cationic moiety of jatrorrhizine or aderivative or analog thereof, and m=1 and n=1.

In certain preferred embodiments, U⁻ is an anionic moiety of obeticholicacid, X⁺ is a cationic moiety of berberine or a derivative or analogthereof, and m=1 and n=1. Exemplary derivatives or analogs of berberineare listed in Table 2. In certain preferred embodiments, U⁻ is ananionic moiety of obeticholic acid, X⁺ is a cationic moiety ofberberine, and m=1 and n=1.

In certain preferred embodiments, U⁻ is an anionic moiety of obeticholicacid, X⁺ is a cationic moiety of metformin or a derivative or analogthereof, and m=1 and n=1. Exemplary derivatives or analogs of metforminare listed in Table 4. In certain preferred embodiments, U⁻ is ananionic moiety of obeticholic acid, X⁺ is a cationic moiety ofmetformin, and m=1 and n=1.

In certain preferred embodiments, U⁻ is an anionic moiety of obeticholicacid, X⁺ is a cationic moiety of carnitine or a derivative or analogthereof, and m=1 and n=1. Exemplary derivatives or analogs of carnitineare listed in Table 5. In certain preferred embodiments, U⁻ is ananionic moiety of obeticholic acid, X⁺ is a cationic moiety ofcarnitine, and m=1 and n=1.

In certain preferred embodiments, is an anionic moiety of obeticholicacid, X⁺ is a cationic moiety of coptisine or a derivative or analogthereof, and m=1 and n=1.

In certain preferred embodiments, U⁻ is an anionic moiety of obeticholicacid, X⁺ is a cationic moiety of palmatine or a derivative or analogthereof, and m=1 and n=1.

In certain preferred embodiments, U⁻ is an anionic moiety of obeticholicacid, X⁺ is a cationic moiety of jatrorrhizine or a derivative or analogthereof, and m=1 and n=1.

TABLE 4 Metformin Derivatives or Analogs

(I) L¹ and L² are independently a bond or —NH—C(NH)—; R¹ is—NR^(1A)R^(1B), substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, or substituted or unsubstituted heteroaryl, whereinR^(1A) and R^(1B) are optionally joined together to form a substitutedor unsubstituted heterocycloalkyl; R² is —NR^(1A)R^(1B), substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, or substituted orunsubstituted heteroaryl, wherein R^(2A) and R^(2B) are optionallyjoined together to form a substituted or unsubstituted heterocycloalkyl;R^(1A), R^(1B), R^(2A), and R^(2B) are independently hydrogen, —OR₄,substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl, orsubstituted or unsubstituted heteroaryl; R³ is hydrogen or unsubstitutedC₁-C₅ alkyl; and R⁴ is hydrogen, substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heterocycloalkyl, substitutedor unsubstituted aryl, or substituted or unsubstituted heteroaryl;

R′ = —H, —Ph, substituted —Ph R = R₁ substituted —Ph R₁ = C₁-C₆ alkyl,C₁-C₄ alkyl, fluorinated alkyl, acyl, ester, aryl, halogen, NO₂, NH₂,—H, —OR₂, —SR₂ R₂ = C₁-C₆ alkyl, C₁-C₄ fluorinated alkyl, acyl

TABLE 5 L-carnitine Derivatives or Analogs

Formula 1 Wherein A is selected from the group consisting of a singlebond. ‘Of. or iCH2i; m and n van independently and are an integer from 1to 15; p and q vary independently from 0 to 1; B is iCR3R4; D isselected from the group consisting of iCOzRs, ADR6, ADCOR7, iSO3R8,iSO2NH2, iOPO(OR9)(OR1O), A)PO(OR9)(NH2), iOPO(OR9)i OiPO(OR1O)(OR11),wherein R1 to R4 are independently selected from C1-C6 alkyl; and R5 toR1 1 are independently selected from the group consisting of hydrogen;C1-C6 alkyl; C3-C6 cycloalkyl; C2-C6 alkenyl; C6 alkynyl; C5-C10 arylunsubstituted or substituted With C1-C6 alkyl, hydroxyl, C1-C6 alkoxyl,1,3-dioxolanyl, cyano, halo, nitro, trihaloalkyl, carboxyl, C1-C6 acyl,C1-C6 hydroxyalkyl, amino, C1-C6 alkylamino, C1-C6 dialkylamino, C1-C6acylamino, C1-C6 alkoxylcarbonyl; C5-C6 ary lalkyl unsubstituted orsubstituted With C1-C6 alkyl, hydroxyl, C1-C6 alkoxyl, 1,3-dioxolanyl,cyano, halo, triha loalkyl, carboxyl, C1-C6 acyl, C1-C6 hydroxyalkyl,amino, C1-C6 alkylamino, C1-C6 dialkylamino, C1-C6 alkoxylcarbonyl;C1-C6 carboxyalkyl; C1-C6 acylamino; C1-C6 sulfonatoalkyl; C1-C6sulfamylalkyl; and C1-C6 phosphonatoalkyl.

Wherein X is an integer between about 0 and 5

In yet another aspect, the invention generally relates to apharmaceutical composition comprising an amount of an acid-base additionsalt having the formula of:(X⁺)_(m)(U⁻)_(n)  (I)wherein

(a) U⁻ is an anionic moiety of ursodeoxycholic acid or a derivative oranalog thereof;

(b) X⁺ is a cationic moiety of a pharmacologically active organic base;and

(c) m and n are integers independently selected from 1, 2, 3, 4, 5 and 6so as to arrive at a charge neutral salt,

effective to treat, prevent, or reduce one or more diseases or disordersselected from fatty liver, NAFLD and NASH, cholestatic liver diseases,graft-versus-host disease of the liver, chronic viral associated liverdiseases, alcohol-related liver diseases, metabolic diseases ordisorders such as pre-diabetes, diabetes, diabetic dyslipidemia,dyslipidemia in statin-intolerant patients, hyperlipidemia, obesity, ora related disease or disorder thereof in a mammal, including a human,and a pharmaceutically acceptable excipient, carrier, or diluent.

In certain preferred embodiments, the pharmaceutical composition of theinvention is used to treat, prevent, or reduce NASH. In certainpreferred embodiments, the pharmaceutical composition of the inventionis used to treat, prevent, or reduce NAFLD. In certain preferredembodiments, the pharmaceutical composition of the invention is used totreat, prevent, or reduce fatty liver. In certain preferred embodiments,the pharmaceutical composition of the invention is used to treat,prevent, or reduce a disease or disorder selected from cholestatic liverdiseases, graft-versus-host disease of the liver, chronic viralassociated liver diseases, alcohol-related liver diseases, metabolicdiseases or disorders such as pre-diabetes, diabetes, diabeticdyslipidemia, dyslipidemia in statin-intolerant patients,hyperlipidemia, or obesity.

In the context of the pharmaceutical composition of the invention, U⁻can be an anionic moiety of any suitable derivative or analog ofursodeoxycholic, for example, selected from Table 3. X⁺ can be acationic moiety of any suitable pharmacologically active organic base.In certain embodiments, for example, the pharmacologically activeorganic base may be selected from the group consisting of berberine,metformin, carnitine, coptisine, palmatine, and jatrorrhizine. Incertain embodiments, X⁺ can also be a cationic moiety of other organicbase that is generally recognized pharmacologically active for one ormore diseases or disorders selected from fatty liver, NAFLD, NASH,cholestatic liver diseases or graft-versus-host disease of the liver,chronic viral associated liver diseases, alcohol-related liver diseases,metabolic diseases or disorders such as pre-diabetes, diabetes, diabeticdyslipidemia, dyslipidemia in statin-intolerant patients,hyperlipidemia, obesity, or a related disease or disorder thereof in amammal, including a human.

In the context of the pharmaceutical composition of the invention, incertain preferred embodiments, U⁻ is an anionic moiety ofursodeoxycholic acid, X⁺ is a cationic moiety of berberine or aderivative or analog thereof, and m=1 and n=1. Exemplary derivatives oranalogs of berberine are listed in Table 2. In certain preferredembodiments, U⁻ is an anionic moiety of ursodeoxycholic acid, X⁺ is acationic moiety of berberine, and m=1 and n=1. In certain preferredembodiments, U⁻ is an anionic moiety of ursodeoxycholic acid, X⁺ is acationic moiety of metformin or a derivative or analog thereof, and m=1and n=1. Exemplary derivatives or analogs of metformin are listed inTable 4. In certain preferred embodiments, U⁻ is an anionic moiety ofursodeoxycholic acid, X⁺ is a cationic moiety of metformin, and m=1 andn=1. In certain preferred embodiments, U⁻ is an anionic moiety ofursodeoxycholic acid, X⁺ is a cationic moiety of coptisine, and m=1 andn=1. In certain preferred embodiments, U⁻ is an anionic moiety ofursodeoxycholic acid, X⁺ is a cationic moiety of palmatine, and m=1 andn=1. In certain preferred embodiments, U⁻ is an anionic moiety ofursodeoxycholic acid, X⁺ is a cationic moiety of jatrorrhizine, and m=1and n=1.

In the context of the pharmaceutical composition of the invention, incertain preferred embodiments, U⁻ is an anionic moiety of obeticholicacid, X⁺ is a cationic moiety of berberine or a derivative or analogthereof, and m=1 and n=1. Exemplary derivatives or analogs of berberineare listed in Table 2. In certain preferred embodiments, U⁻ is ananionic moiety of obeticholic acid, X⁺ is a cationic moiety ofberberine, and m=1 and n=1. In certain preferred embodiments, U⁻ is ananionic moiety of obeticholic acid, X⁺ is a cationic moiety of metforminor a derivative or analog thereof, and m=1 and n=1. Exemplaryderivatives or analogs of metformin are listed in Table 4. In certainpreferred embodiments, U⁻ is an anionic moiety of obeticholic acid, X⁺is a cationic moiety of metformin, and m=1 and n=1. In certain preferredembodiments, U⁻ is an anionic moiety of obeticholic acid, X⁺ is acationic moiety of coptisine, and m=1 and n=1. In certain preferredembodiments, U⁻ is an anionic moiety of obeticholic acid, X⁺ is acationic moiety of palmatine, and m=1 and n=1. In certain preferredembodiments, U⁻ is an anionic moiety of obeticholic acid, X⁺ is acationic moiety of jatrorrhizine, and m=1 and n=1.

In certain preferred embodiments, the pharmaceutical composition furtherincludes a compound selected from the group consisting of vitamin E,omega-3 fatty acids, S-adenosylmethionine, N-acetyl cysteine, silymarin,polyenylphosphatidylcholine, resveratrol or vitamin D.

In yet another aspect, the invention generally relates to a method fortreating, reducing, or preventing a disease or disorder. The methodincludes administering to a subject in need thereof a pharmaceuticalcomposition comprising an amount of an acid-base addition salt havingthe formula of:(X⁺)_(m)(U⁻)_(n)  (I)wherein

(a) U⁻ is an anionic moiety of ursodeoxycholic acid or a derivative oranalog thereof;

(b) X⁺ is a cationic moiety of a pharmacologically active organic base;and

(c) m and n are integers independently selected from 1, 2, 3, 4, 5 and 6so as to arrive at a charge neutral salt,

effective to treat, prevent, or reduce one or more diseases or disordersselected from fatty liver, NAFLD, NASH, cholestatic liver diseases,graft-versus-host disease of the liver, chronic viral associated liverdiseases, alcohol-related liver diseases, metabolic diseases ordisorders such as pre-diabetes, diabetes, diabetic dyslipidemia,dyslipidemia in statin-intolerant patients, hyperlipidemia, obesity, ora related disease or disorder thereof in a mammal, including a human,and a pharmaceutically acceptable excipient, carrier, or diluent.

In certain preferred embodiments, the method is to treat, prevent, orreduce NASH. In certain preferred embodiments, the method is to treat,prevent, or reduce NAFLD. In certain preferred embodiments, the methodis to treat, prevent, or reduce fatty liver. In certain preferredembodiments, the method is to treat, prevent, or reduce a disease ordisorder selected from cholestatic liver diseases, graft-versus-hostdisease of the liver, chronic viral associated liver diseases,alcohol-related liver diseases, metabolic diseases or disorders such aspre-diabetes, diabetes, diabetic dyslipidemia, dyslipidemia instatin-intolerant patients, hyperlipidemia, obesity, or a relateddisease or disorder.

In the context of the method of the invention, U⁻ can be an anionicmoiety of any suitable derivative or analog of ursodeoxycholic, forexample, selected from Table 3. X⁺ can be a cationic moiety of anysuitable pharmacologically active organic base. In certain embodiments,for example, the pharmacologically active organic base may be selectedfrom the group consisting of berberine, metformin, carnitine andcoptisine, palmatine, and jatrorrhizine. In certain embodiments, X⁺ canalso be a cationic moiety of other organic base that is generallyrecognized pharmacologically active for one or more diseases ordisorders selected from fatty liver, NAFLD and NASH, cholestatic liverdiseases, graft-versus-host disease of the liver, chronic viralassociated liver diseases, alcohol-related liver diseases, metabolicdiseases or disorders such as pre-diabetes, diabetes, diabeticdyslipidemia, dyslipidemia in statin-intolerant patients,hyperlipidemia, obesity, or a related disease or disorder thereof in amammal, including a human.

In the context of the method of the invention, in certain preferredembodiments, U⁻ is an anionic moiety of ursodeoxycholic acid, X⁺ is acationic moiety of berberine or a derivative or analog thereof, and m=1and n=1. Exemplary derivatives or analogs of berberine are listed inTable 2. In certain preferred embodiments, U⁻ is an anionic moiety ofursodeoxycholic acid, X⁺ is a cationic moiety of berberine, and m=1 andn=1. In certain preferred embodiments, U⁻ is an anionic moiety ofursodeoxycholic acid, X⁺ is a cationic moiety of metformin or aderivative or analog thereof, and m=1 and n=1. Exemplary derivatives oranalogs of metformin are listed in Table 4. In certain preferredembodiments, U⁻ is an anionic moiety of ursodeoxycholic acid, X⁺ is acationic moiety of metformin, and m=1 and n=1. In certain preferredembodiments, U⁻ is an anionic moiety of ursodeoxycholic acid, X⁺ is acationic moiety of carnitine or a derivative or analog thereof, and m=1and n=1. Exemplary derivatives or analogs of carnitine are listed inTable 5. In certain preferred embodiments, U⁻ is an anionic moiety ofursodeoxycholic acid, X⁺ is a cationic moiety of carnitine, and m=1 andn=1. In certain preferred embodiments, U⁻ is an anionic moiety ofursodeoxycholic acid, X⁺ is a cationic moiety of coptisine, and m=1 andn=1. In certain preferred embodiments, U⁻ is an anionic moiety ofursodeoxycholic acid, X⁺ is a cationic moiety of palmatine, and m=1 andn=1. In certain preferred embodiments, U⁻ is an anionic moiety ofursodeoxycholic acid, X⁺ is a cationic moiety of jatrorrhizine, and m=1and n=1. In certain preferred embodiments, the pharmaceuticalcomposition further includes a compound selected from the groupconsisting of vitamin E, omega-3 fatty acids, S-adenosylmethionine,N-acetyl cysteine, silymarin, polyenylphosphatidylcholine, resveratrolor vitamin D. In certain preferred embodiments, treating, reducing, orpreventing a disease or disorder is by normalizing liver enzyme levelsof the subject.

In the context of the method of the invention, in certain preferredembodiments, U⁻ is an anionic moiety of obeticholic acid, X⁺ is acationic moiety of berberine or a derivative or analog thereof, and m=1and n=1. Exemplary derivatives or analogs of berberine are listed inTable 2. In certain preferred embodiments, U⁻ is an anionic moiety ofobeticholic acid, X⁺ is a cationic moiety of berberine, and m=1 and n=1.In certain preferred embodiments, U⁻ is an anionic moiety of obeticholicacid, X⁺ is a cationic moiety of metformin or a derivative or analogthereof, and m=1 and n=1. Exemplary derivatives or analogs of metforminare listed in Table 4. In certain preferred embodiments, U⁻ is ananionic moiety of obeticholic acid, X⁺ is a cationic moiety ofmetformin, and m=1 and n=1. In certain preferred embodiments, U⁻ is ananionic moiety of obeticholic acid, X⁺ is a cationic moiety of carnitineor a derivative or analog thereof, and m=1 and n=1. Exemplaryderivatives or analogs of carnitine are listed in Table 5. In certainpreferred embodiments, U⁻ is an anionic moiety of obeticholic acid, X⁺is a cationic moiety of carnitine, and m=1 and n=1. In certain preferredembodiments, U⁻ is an anionic moiety of obeticholic acid, X⁺ is acationic moiety of coptisine, and m=1 and n=1. In certain preferredembodiments, U⁻ is an anionic moiety of obeticholic acid, X⁺ is acationic moiety of palmatine, and m=1 and n=1. In certain preferredembodiments, U⁻ is an anionic moiety of obeticholic acid, X⁺ is acationic moiety of jatrorrhizine, and m=1 and n=1. In certain preferredembodiments, the pharmaceutical composition further includes a compoundselected from the group consisting of vitamin E, omega-3 fatty acids,S-adenosylmethionine, N-acetyl cysteine, silymarin,polyenylphosphatidylcholine, resveratrol or vitamin D. In certainpreferred embodiments, treating, reducing, or preventing a disease ordisorder is by normalizing liver enzyme levels of the subject.

Salts of Berberine or Derivatives

The invention further provides salts of berberine and organic acids,pharmaceutical compositions thereof, as well as related methods of theiruse in treating various diseases or disorders.

Salts of berberine includes those with organic acids such asR-(+)-α-lipoic acid, hydroxycitric acid, eicosapentaenoic acid,docosahexaenoic acidursolic acid, corosolic acid, cinnamic acid, cholicacid, obeticholic acid, ursodeoxycholic acid, oleanolic acid, salicylicacid, betulinic acid, chlorogenic acid, caffeic acid, bassic acid,acetyl L-carnitine, S-allyl cysteine sulphoxide, S-methyl cysteinesulfoxide, pantothenic acid, ascorbic acid, retinoic acid, nicotinicacid, and biotin.

In yet another aspect, the invention generally relates to an acid-baseaddition salt in substantially pure form, having the formula of:(B⁺)_(m)(Y⁻)_(n)  (II)wherein

(a) B⁺ is a cationic moiety of berberine or a derivative or analogthereof;

(b) Y⁻ is an anionic moiety of a pharmacologically active organic acid;and

(c) m and n are integers independently selected from 1, 2, 3, 4, 5 and 6so as to arrive at a charge neutral salt.

In certain embodiments of the acid-base addition salt, the berberinederivative or analog is selected from Table 2.

In certain embodiments of the acid-base addition salt, thepharmacologically active organic acid is selected from the groupconsisting of R-(+)-α-lipoic acid, hydroxycitric acid, eicosapentaenoicacid, docosahexaenoic acid, docosapentaenoic acid, ursolic acid,corosolic acid, cinnamic acid, cholic acid, obeticholic acid,ursodeoxycholic acid, oleanolic acid, salicylic acid, betulinic acid,chlorogenic acid, caffeic acid, bassic acid, acetyl L-carnitine, 5-allylcysteine sulphoxide, S-methyl cysteine sulfoxide, pantothenic acid,ascorbic acid, retinoic acid, nicotinic acid, biotin and other organicacid that is generally recognized pharmacologically active for one ormore diseases or disorders selected from metabolic disorders, heartdiseases, atherosclerosis, neurodegenerative diseases, liver diseases,sarcopenia, muscle atrophy, inflammation, and cancer, or a relateddisease or disorder thereof in a mammal, including a human by those ofskill in the art.

In certain embodiments, B⁺ is a cationic moiety of berberine and Y⁻ isan anionic moiety of R-(+)-α-lipoic acid, and m=1 and n=1.

In certain embodiments, B⁺ is a cationic moiety of berberine and Y⁻ isan anionic moiety of hydroxycitric acid, and m=1 and n=1, or m=2, n=1,or m=3, n=1.

In certain embodiments, B⁺ is a cationic moiety of berberine and Y⁻ isan anionic moiety of EPA, and m=1 and n=1.

In certain embodiments, B⁺ is a cationic moiety of berberine and Y⁻ isan anionic moiety of DHA, and m=1 and n=1.

In certain embodiments, B⁺ is a cationic moiety of berberine and Y⁻ isan anionic moiety of DPA, and m=1 and n=1.

In certain embodiments, B⁺ is a cationic moiety of berberine and Y⁻ isan anionic moiety of ursolic acid, and m=1 and n=1.

In certain embodiments, B⁺ is a cationic moiety of berberine and Y⁻ isan anionic moiety of corosolic acid, and m=1 and n=1.

In certain embodiments, B⁺ is a cationic moiety of berberine and Y⁻ isan anionic moiety of cholic acid, and m=1 and n=1.

In certain embodiments, B⁺ is a cationic moiety of berberine and Y⁻ isan anionic moiety of ursodeoxycholic acid, and m=1 and n=1.

In certain embodiments, B⁺ is a cationic moiety of berberine and Y⁻ isan anionic moiety of obeticholic acid, and m=1 and n=1.

In yet another aspect, the invention generally relates to apharmaceutical composition comprising an amount of an acid-base additionsalt having the formula of:(B⁺)_(m)(Y⁻)_(n)  (II)wherein

(a) B⁺ is a cationic moiety of berberine or a derivative or analogthereof;

(b) Y⁻ is an anionic moiety of a pharmacologically active organic acid;and

(c) m and n are integers independently selected from 1, 2, 3, 4, 5 and 6so as to arrive at a charge neutral salt,

effective to treat, prevent, or reduce one or more diseases or disordersselected from metabolic disorders, heart diseases, atherosclerosis,neurodegenerative diseases, liver diseases, sarcopenia, muscle atrophy,inflammation, and cancer, or a related disease or disorder thereof in amammal, including a human, and a pharmaceutically acceptable excipient,carrier, or diluent.

In certain embodiments, the disease or disorder is a metabolic disorderwhich is selected from diabetes, diabetic complications, dyslipidemia,diabetic dyslipidemia, dyslipidemia in statin-intolerance patients,hypercholesterolemia, hypertriglyceridemia, metabolic syndromes andpre-diabetes. In certain embodiments, the metabolic disorder is type 1or type 2 diabetes.

In certain embodiments, the disease or disorder is cancer. In certainembodiments, the cancer is selected from the group consisting of breastcancer, prostate cancer, lung cancer, hepatocellular carcinoma,pancreatic cancer, gastric carcinoma, colorectal cancer, leukemia,multiple myeloma, melanoma and glioblastoma.

In certain embodiments, the disease or disorder is heart diseases.

In certain embodiments, the disease or disorder is atherosclerosis.

In certain embodiments, the disease or disorder is sarcopenia.

In certain embodiments, the disease or disorder is muscle atrophy. Incertain embodiments, the disease or disorder is muscle atrophy which isselected from skeletal muscle atrophy.

In certain embodiments of the pharmaceutical composition, the berberinederivative or analog is selected from Table 2.

In certain embodiments of the pharmaceutical composition, thepharmacologically active organic acid is selected from the groupconsisting of R-(+)-α-lipoic acid, hydroxycitric acid, eicosapentaenoicacid, docosahexaenoic acid, docosapentaenoic acid, ursolic acid, andcorosolic acid, cinnamic acid, cholic acid, obeticholic acid,ursodeoxycholic acid, oleanolic acid, salicylic acid, betulinic acid,chlorogenic acid, caffeic acid, bassic acid, acetyl L-carnitine, S-allylcysteine sulphoxide, S-methyl cysteine sulfoxide, pantothenic acid,ascorbic acid, retinoic acid, nicotinic acid, biotin and other organicacid that is generally recognized pharmacologically active for one ormore diseases or disorders selected from metabolic disorders, heartdiseases, atherosclerosis, neurodegenerative diseases, liver diseases,sarcopenia, muscle atrophy, inflammation, and cancer, or a relateddisease or disorder thereof in a mammal, including a human by those ofskill in the art.

In certain embodiments, B⁺ is a cationic moiety of berberine and X⁻ isan anionic moiety of R-(+)-α-Lipoic acid, and m=1 and n=1. In certainembodiments, B⁺ is a cationic moiety of berberine and X⁻ is an anionicmoiety of hydroxycitric acid, and m=1 and n=1, or m=2, n=1, or m=3, n=1.In certain embodiments, B⁺ is a cationic moiety of berberine and X⁻ isan anionic moiety of EPA, and m=1 and n=1. In certain embodiments, B⁺ isa cationic moiety of berberine and X⁻ is an anionic moiety of DHA, andm=1 and n=1. In certain embodiments, B⁺ is a cationic moiety ofberberine and X⁻ is an anionic moiety of DPA, and m=1 and n=1. Incertain embodiments, B⁺ is a cationic moiety of berberine and X⁻ is ananionic moiety of ursolic acid, and m=1 and n=1.

In yet another aspect, the invention generally relates to a method fortreating, reducing, or preventing a disease or disorder. The methodincludes administering to a subject in need thereof a pharmaceuticalcomposition comprising an amount of an acid-base addition salt havingthe formula of:(B⁺)_(m)(Y⁻)_(n)  (II)wherein

(a) B⁺ is a cationic moiety of berberine or a derivative or analogthereof;

(b) Y⁻ is an anionic moiety of a pharmacologically active organic acid;and

(c) m and n are integers independently selected from 1, 2, 3, 4, 5 and 6so as to arrive at a charge neutral salt,

effective to treat, prevent, or reduce one or more diseases or disordersselected from metabolic disorders, heart diseases, atherosclerosis,neurodegenerative diseases, liver diseases, sarcopenia, muscle atrophy,inflammation, and cancer, or a related disease or disorder thereof in amammal, including a human, and a pharmaceutically acceptable excipient,carrier, or diluent

In certain embodiments, the metabolic disorder is selected fromdiabetes, diabetic complications, dyslipidemia, diabetic dyslipidemia,dyslipidemia in statin-intolerant patients, obesity, metabolicsyndromes, pre-diabetes, fatty liver, NAFLD, and NASH. In certainembodiments, the metabolic disorder is type 1 or type 2 diabetes.

In certain embodiments, the berberine derivative or analog is selectedfrom Table 2.

In certain embodiments, the pharmacologically active organic acid isselected from the group consisting of R-(+)-α-lipoic acid, hydroxycitricacid, eicosapentaenoic acid, docosahexaenoic acid, docosapentaenoicacid, ursolic acid, and corosolic acid, cinnamic acid, cholic acid,obeticholic acid, ursodeoxycholic acid, oleanolic acid, salicylic acid,betulinic acid, chlorogenic acid, caffeic acid, bassic acid, acetylL-carnitine, S-allyl cysteine sulphoxide, S-methyl cysteine sulfoxide,pantothenic acid, ascorbic acid, retinoic acid, nicotinic acid, biotinand other organic acid that is generally recognized pharmacologicallyactive for one or more diseases or disorders selected from metabolicdisorders, heart diseases, atherosclerosis, neurodegenerative diseases,liver diseases, sarcopenia, muscle atrophy, inflammation, and cancer, ora related disease or disorder thereof in a mammal, including a human bythose of skill in the art.

In certain embodiments of the method, B⁺ is a cationic moiety ofberberine and Y⁻ is an anionic moiety of R-(+)-α-lipoic acid, and m=1and n=1, and the subject suffers from diabetes and diabeticcomplications.

In certain embodiments of the method, B⁺ is a cationic moiety ofberberine and Y⁻ is an anionic moiety of hydroxycitric acid, and m=1 andn=1, or m=2, n=1, or m=3, n=1, and the subject suffers from diabetes andobesity.

In certain embodiments of the method, B⁺ is a cationic moiety ofberberine and Y⁻ is an anionic moiety of EPA, and m=1 and n=1, and thesubject suffers from diabetes and dyslipidemia, or heart diseases,atherosclerosis, or neurodegenerative diseases.

In certain embodiments of the method, B⁺ is a cationic moiety ofberberine and Y⁻ is an anionic moiety of DHA, and m=1 and n=1, and thesubject suffers from diabetes and dyslipidemia, or heart diseases,atherosclerosis, or neurodegenerative diseases.

In certain embodiments of the method, B⁺ is a cationic moiety ofberberine and Y⁻ is an anionic moiety of DPA, and m=1 and n=1, and thesubject suffers from diabetes and dyslipidemia, or heart diseases,atherosclerosis, or neurodegenerative diseases.

In certain embodiments of the method, B⁺ is a cationic moiety ofberberine and Y⁻ is an anionic moiety of ursolic acid, and m=1 and n=1,and the subject suffers from diabetes and sarcopenia, or muscle atrophy.

In certain embodiments of the method, B⁺ is a cationic moiety ofberberine and Y⁻ is an anionic moiety of corosolic acid, and m=1 andn=1, and the subject suffers from diabetes and sarcopenia, or muscleatrophy.

In certain embodiments of the method, B⁺ is a cationic moiety ofberberine and Y⁻ is an anionic moiety of cholic acid, and m=1 and n=1,and the subject suffers from dyslipidemia, fatty liver, NAFLD or NASH.

In certain embodiments of the method, B⁺ is a cationic moiety ofberberine and Y⁻ is an anionic moiety of obeticholic acid, and m=1 andn=1, and the subject suffers from dyslipidemia, fatty liver, NAFLD orNASH.

In certain embodiments of the method, B⁺ is a cationic moiety ofberberine and Y⁻ is an anionic moiety of ursodeoxycholic acid, and m=1and n=1, and the subject suffers from dyslipidemia, fatty liver, NAFLDor NASH.

In certain preferred embodiments of the method, treating, reducing, orpreventing a metabolic disorder is by reducing blood glucose levels ofthe subject. In certain preferred embodiments of the method, treating,reducing, or preventing a metabolic disorder is by reducing totalcholesterol (TC), triglyceride (TG) and low-density lipoproteincholesterol (LDL-c) levels, increasing high-density lipoproteincholesterol (HDL-c) levels of the subject. In certain preferredembodiments of the method, treating, reducing, or preventing a metabolicdisorder is by normalizing liver enzyme levels of the subject. Incertain preferred embodiments of the method, treating, reducing, orpreventing a metabolic disorder is by normalizing liver lipid levels ofthe subject. In certain preferred embodiments of the method, treating,reducing, or preventing a metabolic disorder is by altering insulinsignaling pathway such that glucose levels are reduced. In certainpreferred embodiments of the method, treating, reducing, or preventing ametabolic disorder is by regulating multiple metabolic pathways such asincreasing secretion of insulin, improving insulin sensitivity, reducinggluconeogenesis in liver, reducing glucose absorption, amelioratingdyslipidemia, anti-inflammation to achieve the desired pharmacologicaleffects.

The following examples are meant to be illustrative of the practice ofthe invention, and not limiting in any way.

EXAMPLES Example 1. Efficacy of the Combination of Berberine andEicosapntemacnioc Acid (EPA), Docosahexaenoic Acid (DHA); or Berberineand Ursolic Acid (UA) in High Fat Diet/Streptozocin Induced DiabeticMice Model

This example describes an in vivo efficacy study of the combinationsdisclosed in the present invention using a high fat diet (HFD) andstreptozotocin (STZ) induced diabetic mice model.

Sixty NIH male mice of 4 weeks old were acquired from GuangzhouInstitute of Laboratory Animal. After acclimatization for one week, fivemice were selected as normal control group (Group 1), the restfifty-five mice were administered single dose of STZ at the dose of 40mg/kg, and fed with HFD (in which 40% of calories is from fat) for 7days to establish a diabetic animal model resembling the pathophysiologyof type 2 diabetes in human. Mice in the normal control group were notadministered STZ, and fed with normal chow diet.

For the fifty-five mice induced with STZ and HFD, forty of them withfasting blood glucose greater than 12.0 mmol/L at the seventh day postof STZ administration were selected and randomized into 4 groups (n=10per group):

Group 2: Vehicle control (Normal saline)

Group 3: Positive control (Metformin 300 mg/kg)

Group 4: Combination of berberine (150 mg/kg) and UA (150 mg/kg)

Group 5: Combination of berberine (150 mg/kg), EPA (75 mg/kg) and DHA(75 mg/kg)

The mice from Group 2 to 5 were treated with the corresponding testingarticles indicated above once daily by intragastric gavage. HFDcontinued throughout the duration of the treatment of 28 days. Thenormal mice (Group 1) were treated with normal saline by intragastricgavage. Fasting blood glucose, total cholesterol (TC) and triglyceride(TG) levels, food intake, water intake and body weight were measuredthroughout the study.

On day 28 of the treatment, an oral glucose tolerance test (OGTT) wasperformed in 12-hour fasted animals. For the OGTT, after the measurementof the basal glucose concentration (T=−30 min), mice received an oralglucose challenge at 2.5 g/kg and glucose values were determined byglucometer (ACCU-CHEK Active, Roche) at 0, 30, 60 and 120 min.

After OGTT, blood samples were collected for the measurement of bloodglucose, TC and TG. The mice were sacrificed, and the pancreas, liver,kidney and fat were harvested for histopathology analysis.

The experimental results were listed in Table 6 and Table 7.

TABLE 6 Average body weight, food intake and water intake in differenttreatment group* Weight (g) Food Intake (g) Water intake (mL) Group Day0 Day 27 Day 3 Day 27 Day 3 Day 6 Day 27 No. 1 Normal 27.80 ± 1.45 34.20± 2.84 3.69 4.88  7.60  8.40  7.20 (n = 5) (n = 5) (n = 5) (n = 5) (n =5) (n = 5) (n = 5) No. 2 Vehicle 27.20 ± 1.47 34.70 ± 3.32 7.46 13.78 19.20 24.40 32.00 control (n = 10) (n = 10) (n = 10) (n = 10) (n = 10)(n = 10) (n = 10) No. 3 Metformin 26.30 ± 2.54 36.99 ± 3.90 8.66 9.3719.40 23.60 34.40 (n = 10) (n = 10) (n = 10) (n = 10) (n = 10) (n = 10)(n = 10) No. 4 Berberine + 26.20 ± 1.77 26.13 ± 2.95 9.08 6.59 19.4019.00 13.25 UA (n = 10) (n = 8)^(a,b) (n = 10) (n = 8)^(e) (n = 10) (n =10) (n = 8)^(a,b) No. 5 Beberine + 25.90 ± 2.04 29.76 ± 4.89 8.35 5.7919.40 20.00 14.20 EPA/DHA (n = 10) (n = 10)^(c,d) (n = 10) (n = 10)^(f)(n = 10) (n = 10) (n = 10)^(c,d) *1) Body weight was measured on day 0,and on day 27 the day prior to the sacrifice, to minimize the variationscaused by 12-hour fasting. 2) Food intake and water intake were measuredtwice weekly throughout the study, representative data were presentedhere. 3) Two animals in Group 4 were found dead throughout the study.The autopsy results indicated inappropriate handling when dosing byintra-gavage. ^(a,b)significant difference between G4 and G2, G3 (p <0.001) ^(c,d)significant difference between G5 and G2, G3 (p < 0.01)^(e)significant difference between G4 and G2 (p < 0.05) ^(f)significantdifference between G5 and G2 (p < 0.01)

TABLE 7 Average fasting blood glucose, total cholesterol andtriglyceride in different treatment group Fasting blood glucose TotalCholesterol Triglyceride (mmol/L) (mg/dL) (mmol/L) Group Day 0 Day 28Day 0 Day 28 Day 0 Day 28 No. 1 Normal 11.59 ± 1.12  3.84 ± 0.26 222.93± 16.17 234.63 ± 57.65 1.02 ± 0.31 1.94 ± 0.33 (n = 5) (n = 5) (n = 5)(n = 5) (n = 5) (n = 5) No. 2 Vehicle 24.74 ± 8.47 24.58 ± 6.01 303.90 ±65.51  335.49 ± 103.95 2.16 ± 0.78 4.99 ± 6.01 control (n = 10) (n = 10)(n = 10) (n = 10) (n = 10) (n = 10) No. 3 Metformin 24.08 ± 5.44 21.66 ±4.71 297.96 ± 67.09  436.99 ± 159.73 2.40 ± 1.03 6.06 ± 6.71 (n = 10) (n= 10) (n = 10) (n = 10) (n = 10) (n = 10) No. 4 Berberine + 23.78 ± 8.5614.70 ± 7.22 327.52 ± 55.60 264.30 ± 81.49 2.15 ± 0.87 2.87 ± 1.28 UA (n= 10) (n = 8)^(a) (n = 10) (n = 8)^(b) (n = 10) (n = 8) ^(d) No. 5Beberine + 24.36 ± 7.43 18.20 ± 8.71 303.07 ± 47.27 242.39 ± 53.82 2.56± 1.01 4.16 ± 3.66 EPA/DHA (n = 10) (n = 10) (n = 10) (n = 10)^(c) (n =10) (n = 10) ^(a)significant difference between G4 and G2 (p < 0.01)^(b,c)significant difference between G4, G5 and G3 (p < 0.001) ^(d)significant difference between G4 and G3 (p < 0.05)

These results demonstrated that the combinations of berberine andEPA/DHA, and berberine and UA effectively ameliorated symptoms ofdiabetes HFD/STZ induced diabetic mice model. In contrast, although usedat the suggested therapeutic dose, metformin, an oral anti-diabetic drugused as the first-line of choice for the treatment of type 2 diabetesdid not demonstrate a clear efficacy in this study, further studies arebeing conducted to verify the observations made in this study.

Example 2. Synergistic Effects of the Combination of Berberine andHydroxycitric Acid in a High Fat Diet Induced Obesity Mice Model

This example describes an in vivo efficacy study of the combinationsdisclosed in the present invention using a high fat diet (HFD) inducedobesity mice model.

Fifty NIH male mice of 4 weeks old were acquired (Guangzhou Institute ofLaboratory Animal). After acclimatization for one week, eight mice wereselected as normal control; the rest forty-two mice were fed with HFD(in which 40% of calories is from fat) for 14 days to establish a HFDinduced obesity mice model resembling the pathophysiology of metabolicsyndrome in human. Normal control mice were fed with normal chow diet.For the forty-two mice fed with HFD for 14 days, thirty-two of them withbody weight 15-20% above the normal control mice were selected andrandomized into 4 groups (n=8 per group):

Group 1: Vehicle control (1% Carboxymethyl Cellulose (CMC) solution)

Group 2: Berberine (50 mg/kg in 1% CMC solution)

Group 3: Hydroxycitric acid (50 mg/kg in 1% CMC solution)

Group 4: Combination (berberine (50 mg/kg), and hydroxycitric acid (50mg/kg) in 1% CMC solution)

The mice from Group 1 to 4 were treated with the corresponding testingarticles indicated above once daily by intragastric gavage. HFDcontinued throughout the duration of the treatment of 28 days. Bloodglucose (fasting and non-fasting), total cholesterol (TC) andtriglyceride (TG) levels, food intake, water intake and body weight weremeasured throughout the study.

On day 28 of the treatment, an oral glucose tolerance test (OGTT) wasperformed in 12-hour fasted animals. For the OGTT, after the measurementof the basal glucose concentration (T=−30 min), mice received an oralglucose challenge at 2.5 g/Kg and glucose values were determined byglucometer (ACCU-CHEK Active, Roche) at 0, 30, 60 and 120 min.

After OGTT, blood samples were collected for the measurement of bloodglucose, TC and TG. The mice were sacrificed, and the pancreas, liver,kidney and fat were harvested for histopathology analysis.

The study has been carried out for 15 days, and the interim experimentalresults were presented in FIGS. 1-4.

These results demonstrated that the trend of synergistic effects of theberberine and hydroxycitric acid combination. In particular, when usingindividually at the dose of 50 mg/Kg, neither berberine (Group 2), norhydroxycitric acid (Group 3) demonstrated any pharmacological effectscomparing to the vehicle control group (Group 1); however, when usingtogether (Group 4), reduction in the body weight gain and normalizationof the blood glucose level were observed. Additional data will becollected upon the completion of the study.

Example 3. Synthesis and Analysis of Metformin Ursodeoxycholate Salt

5 mmol metformin hydrochloride was dissolved in NaOH aqueous solutionand allowed to react at room temperature until a clear colorlesssolution was obtained. The solvent was evaporated to yield a whitepowder. The white powder was added into absolute ethanol and then theobtained suspension was filtered to remove white precipitate (NaCl). Thefiltrate was rotary evaporated and then dried in vacuo to yield a whitepowder of Met-OH. The Met-OH was dissolved in absolute ethanol and wasreacted with UDCA at room temperature to yield a clear, light yellowsolution. The solution was rotary evaporated and the residue dried undervacuum (room temp). The resulting white powder was then characterizedwith ¹H NMR and IR (FIGS. 5-6), which indicated the formation ofmetformin ursodeoxycholate salt with 1:1 stoichiometry of Met:UDCA.

Example 4. Synthesis and Analysis of Berberine Ursodeoxycholate Salt

BBR-Cl (1.0 eq) was dissolved into hot distilled water and then thereaction mixture was cooled to room temperature. At the same time,ursodeoxycholic acid (0.9-1.5 eq.) was dissolved into anhydrous ethanol,The aqueous solution of NaCO₃ (0.9-1.5 eq) was added drop wise into theobtained ethanol solution of ursodeoxycholic acid. Then the resultingreaction mixture was stirred for 15-45 minutes and an ursodeoxycholicacid sodium salt solution was obtained.

The BBR-Cl solution was added drop wise to the above ursodeoxycholicacid sodium salt solution under 60-80° C. The mixture was allowed tostir at the same temperature for 2 hours, and then was cooled to roomtemperature. The precipitated solid was filtered and the wet cake wascollected and dried under vacuum below a temperature of 40° C. toproduce crude berberine ursodeoxycholate.

Crude berberine ursodeoxycholate was purified through crystallizationwith ethanol and ethyl acetate. The mixture was allowed to stir for 7-8hours, and then was centrifuged to remove solvent and collect yellowpowder. The yellow powder was rinsed with ethyl acetate again andrepeated the above procedures twice. The final resulted yellow powderwas dried under vacuum at a temperature of 40° C. to obtain purifiedberberine ursodeoxycholate.

The resulting yellow powder was then characterized with ¹H-NMR, IR, andMS (FIG. 7-12). From the ¹H-NMR (FIG. 7-8) and IR (FIG. 9-10) cleardistinctions between the simple mixture of berberine and UDCA (1:1)versus berberine ursodeoxycholate can be seen which indicated theformation of berberine ursodeoxycholate salt with 1:1 stoichiometry ofBBR:UDCA. The MS spectra (FIG. 11-12) indicated that in negative MSmode, molecular mass of UDCA [M-H]⁻ 391.28 was identified. And inpositive MS mode, molecular mass of BBR⁺ 336.14 was identified.

An alternate synthetic method exploits the high EtOH solubility ofberberine ursodeoxycholate coupled with the solubility of BBR in MeOHand the solubility of UDCA sodium in EtOH. For example:

1) Dissolve BBR (1.5 eq) in MeOH at RT. (Solution A)

2) Dissolve UDCA (0.9-1.5 eq) in EtOH at RT, add sodium ethoxidesolution (Solution B)

3) Add Solution A to Solution B at RT and stir for 2-5 hrs. Remove NaClby vacuum filtration, and concentrate filtrate (T<40° C.).

4) Purify crude berberine ursodeoxycholate by dissolving the crudeproduct in EtOH (or other suitable solvent) and removing residual NaClby filtration. Alternately, it may be possible to purify the crudeproduct by “crystallization” from a suitable solvent.

Example 5. Efficacy of Berberine Ursodeoxycholate (BUDCA) in High FatDiet Induced Non-Alcoholic Fatty Liver Mice Model

This example describes an in vivo efficacy study of BUDCA disclosed inthe present invention using a high fat diet (HFD) induced non-alcoholicfatty liver mice model.

91 NIH male mice of 4 weeks old were obtained from Vital RiverLaboratories (Beijing, China). After acclimatization for one week, 13mice were selected as control group (Group 1, G1) with normal chow dietfed, and the other 78 mice were fed with HFD (in which 40% of caloriesis from fat) for 4 weeks to establish an animal model resembling thepathophysiology of non-alcoholic fatty liver in human.

Following 4 weeks of high fat dietary intervention, the 78 mice weredivided into 6 groups according to body weight (n=13 per group):

Group 2, G2: Vehicle control (0.5% CMC-Na solution)

Group 3, G3: Low-dose group of BUDCA (30 mg/kg)

Group 4, G4: Middle-dose group of BUDCA (100 mg/kg)

Group 5, G5: High-dose group of BUDCA (300 mg/kg)

Group 6, G6: BBR control group (Berberine HCl, 150 mg/Kg)

Group 7, G7: UDCA control group (Ursodeoxycholic acid, 150 mg/Kg)

The mice from G2 to G7 were administered with the corresponding testingarticles indicated above once daily by intragastric gavage. HFDcontinued throughout the duration of the treatment of 6 weeks. Thenormal mice (G1) were treated with vehicle (0.5% CMC-Na solution) viaintragastric gavage. At the end of experiment, the following biochemicalparameters were measured and tests were carried out:

-   -   Body weight, the weight ratio of liver    -   Total cholesterol (TC) and triglyceride (TG) levels, high        density lipoprotein cholesterol (HDL-C) and low density        lipoprotein cholesterol (LDL-C) levels    -   Alanine aminotransferase (ALT) and aspartate aminotransferase        (AST) levels    -   Superoxide dismutase (SOD) activity and malondialdehyde (MDA)        level    -   Oral glucose tolerance test (OGTT)    -   Histopathological examination of liver (Sultan III staining)

After 6 weeks treatment, blood was collected via retro-orbital bleedingof each 12-hour fasted animal. Liver was harvested by surgery forhistopathology analysis after weight measurement. Then the serum wasisolated for the determination of TC, TG, HDL-C, LDL-C, ALT, AST, SODand MDA.

One week before sacrifice (Week 5 of treatment), an oral glucosetolerance test (OGTT) was performed in 12-hour fasted animals. For theOGTT, all mice received an oral glucose challenge at 2.0 g/Kg and theblood glucose concentrations were determined by glucometer (ACCU-CHEKActive, Roche) at 0, 30, 60, 90 and 120 min.

The liver tissue was histopathologically evaluated by Sultan IIIstaining after frozen section.

The experimental results were listed as following.

TABLE 8 Body weight and biochemical parameters in various groups Bodyweight Weight ratio TC TG HDL-C LDL-C GROUP (g) of liver (%) (mmol/L)(mmol/L) (mmol/L) (mmol/L) G1 42.6 ± 0.82  3.77 ± 0.12 4.76 ± 0.18  0.75± 0.07  2.74 ± 0.11 0.61 ± 0.05 G2 45.7 ± 0.68**  4.41 ± 0.15**  7.60 ±0.76** 2.03 ± 0.19** 2.65 ± 0.26  1.79 ± 0.24** G3 40.3 ± 1.18## 4.33 ±0.13 5.49 ± 0.25# 1.18 ± 0.20## 2.33 ± 0.15  1.05 ± 0.09## G4 41.7 ±1.59#  4.15 ± 0.09 5.28 ± 0.50# 0.92 ± 0.06## 2.26 ± 0.22  1.06 ± 0.11#G5 41.5 ± 1.16## 4.27 ± 0.10 5.95 ± 0.54  0.85 ± 0.06## 2.48 ± 0.15 1.03 ± 0.22# G6 42.4 ± 1.37#  4.26 ± 0.12 5.97 ± 0.47  1.00 ± 0.08##2.47 ± 0.20 1.42 ± 0.21 G7 42.0 ± 1.16#  4.77 ± 0.26 4.77 ± 0.70# 1.20 ±0.22#  2.13 ± 0.33 1.37 ± 0.26 Data are expressed as the mean ± S.E.M (n= 7~13). *p < 0.05, **p < 0.01 G2 vs. G1 #p < 0.05, ##p < 0.01 G3, G4,G5, G6, or G7 vs. G2

TABLE 9 Liver function in various groups GROUP ALT (U/L) AST (U/L) G132.7 ± 2.88 154.6 ± 10.01 G2 37.4 ± 7.28  250.4 ± 36.73* G3 30.6 ± 4.37148.3 ± 7.15# G4 29.0 ± 3.95  140.2 ± 16.32# G5 27.8 ± 3.08  163.5 ±11.63# G6 37.1 ± 4.08 198.7 ± 18.93 G7 30.6 ± 5.73 162.86 ± 29.42  Dataare expressed as the mean ± S.E.M (n = 7~13). *p < 0.05 G2 vs. G1 #p <0.05 G3, G4, or G5 vs. G2

TABLE 10 Oxidative stress index in various groups GROUP SOD (U/mL) MDA(mmol/L) G1 84.53 ± 5.64  5.67 ± 0.70  G2  38.23 ± 11.61** 24.11 ±6.50** G3 61.05 ± 11.59 12.34 ± 2.89  G4  91.83 ± 4.90## 8.02 ± 1.08# G5 97.54 ± 4.88## 7.78 ± 1.66# G6 77.03 ± 8.98# 9.30 ± 2.14# G7 44.75 ±11.99 18.94 ± 4.42  Data are expressed as the mean ± S.E.M (n = 7~13).**p < 0.01 G2 vs. G1 #p < 0.05, ##p < 0.01 G4, G5, or G6 vs. G2

These results demonstrated that the ionic compound berberineursodeoxycholate dose-dependently ameliorated symptoms of non-alcoholicfatty liver in the mice model induced by HFD. In contrast, although usedat the typically suggested therapeutic dose, berberine orursodeoxycholic acid monotherapy was not as effective as berberineursodeoxycholate. It was also noted that, at the time of sacrifice, nogastrointestinal side effects were observed in berberineursodeoxycholate treated animals, whereas 50% of animals in theberberine treatment group showed gastrointestinal side effects.

Example 6. Efficacy of BUDCA in High Fat Diet-Induced Fatty Liver GoldenHamster Model

This example describes an in vivo efficacy study of BUDCA salt disclosedin the present invention using a high fat diet (HFD) induced fatty livergolden hamster model.

Forty-two SPF golden hamsters with the body weight of 90-100 g wereacquired from Vital River Laboratory Animal Technology Co., Ltd. Afteracclimatization for one week, eight hamsters were selected as normalcontrol group (Group 1), which were fed with normal chow diet. The restthirty-four hamsters were fed with HFD for two weeks to establish afatty liver animal model resembling the pathophysiology of NAFLD inhuman.

For the thirty-four hamsters induced with HFD, twenty-four of them withTC level of 17.96±1.70 mmol/L at the fourteenth day post of HFD wereselected and randomized into three groups (n=8 per group):

Group 2: Model control (0.5% tragacanth solution 10 ml/kg);

Group 3: Low-dose (BUDCA 50 mg/kg);

Group 4: High-dose (BUDCA 200 mg/kg)

The hamsters from Group 2 to 4 were treated with corresponding testingarticles indicated above once a day by intragastric gavage, and HFDcontinued throughout the duration of the 7-week administration. Thenormal hamsters in Group 1 were treated with 0.5% tragacanth solution(10 ml/kg) by intragastric gavage. Serum lipid and blood glucose level,liver function index, food intake and body weight were measuredthroughout the study. After the 7-week administration, the hamsters weresacrificed and dissected for the general observation andhistopathological analysis of liver tissue. The experimental resultswere shown as following:

Food Intake and Body Weight:

There is no significant difference on food intake between the modelcontrol group and the medicated groups (P>0.05). The body weight of thehigh-dose group significantly reduced in the first week of treatment(P<0.05) and no significant changes were observed in the rest time oftreatment (P>0.05). No significant changes were observed in the bodyweight of the low-dose group throughout the study (P>0.05).

Serum Lipid and Blood Glucose Level:

In the model control group, the TC level, TG level, LDL-c level, valueof TC/HDL-c and arterial stiffness index (AI) were increasedsignificantly comparing with those of the normal control group (P<0.01),and the compensatory rising of HDL-c level was observed significantlytoo (P<0.01) while there was no significant difference in blood glucoselevel between the model control group and the normal control group(P>0.05). Comparing with the model control group, the TC level of bothlow-dose group and high-dose group were significantly declined (P<0.01),and the decreasing amplitude in high-dose group was greater than that inlow-dose group (Table 11).

TABLE 11 The Effect of BUDCA on Serum TC Level of HyperlipidemicHamsters (mmol · L⁻¹, X ± s, n = 8) Serum TC Treatment Period GroupDosage level on Day 0 Week 1 Week 3 Week 5 Week 7 Group 1 10 ml · kg⁻¹0.5%  4.17 ± 0.30 3.03 ± 0.94  4.14 ± 0.32  4.21 ± 0.34  4.05 ± 0.33 tragacanth solution Group 2 10 ml · kg⁻¹, 0.5%   17.56 ± 2.59ΔΔ 15.98 ±2.93ΔΔ  14.21 ± 4.56ΔΔ  17.01 ± 4.65ΔΔ 21.74 ± 6.44ΔΔ  tragacanthsolution Group 3 50 mg · kg⁻¹ BUDCA 18.14 ± 1.13 9.33 ± 1.52** 7.58 ±2.01** 10.50 ± 2.89**  9.78 ± 2.58** Group 4 200 mg · kg⁻¹ BUDCA 18.18 ±1.10 7.51 ± 0.71** 6.75 ± 1.00**  5.38 ± 1.24** 4.95 ± 0.84** Note:Comparing with the normal control group, ^(Δ) = P < 0.05, ΔΔ= P < 0.01;Comparing with the model control group, * = P < 0.05, **= P < 0.01.

Comparing with the model control group, the TG level of low-dose groupin Week 1 and the TG level of high-dose group in Week 1 and Week 7 weresignificantly declined (P<0.01), and the decreasing amplitude inhigh-dose group was greater than that in low-dose group too (Table 12).

TABLE 12 The Effect of BUDCA on Serum TG Level of HyperlipidemicHamsters (mmol · L⁻¹, X ± s, n = 8) Serum TG Treatment Period GroupDosage level on Day 0 Week 1 Week 3 Week 5 Week 7 Group 1 10 ml · kg⁻¹2.21 ± 0.27 2.04 ± 0.85  1.13 ± 0.27 1.47 ± 0.47 1.07 ± 0.20 0.5%tragacanth solution Group 2 10 ml · kg⁻¹,   5.87 ± 1.38ΔΔ  5.77 ± 1.17ΔΔ  2.74 ± 0.94Δ   3.98 ± 1.35ΔΔ   4.79 ± 2.21ΔΔ 0.5% tragacanth solutionGroup 3 50 mg · kg⁻¹ BUDCA 5.97 ± 1.19 3.60 ± 0.78** 2.98 ± 1.31 4.51 ±3.10 3.88 ± 1.21 Group 4 200 mg · kg⁻¹ BUDCA 6.31 ± 1.75 3.00 ± 0.67**2.68 ± 1.09 3.04 ± 1.68  1.90 ± 0.66** Note: Comparing with the normalcontrol group, Δ= P < 0.05, ΔΔ= P < 0.01; Comparing with the modelcontrol group, * = P < 0.05, **= P < 0.01

Comparing with the model control group, the serum LDL-c level, the valueof TC/HDL-c and the AI value of both high-dose group and low-dose groupwere significantly decreased (P<0.01), and the HDL-c level of high-dosegroup was significantly declined too (P<0.01). Moreover, the serum LDL-clevel, value of TC/HDL-c and AI value of high-dose group were verysimilar to those of normal control group after 7-week administration(FIG. 16).

Liver Function Index:

In the model control group, the serum AST and ALT level weresignificantly risen comparing with those of the normal control group(P<0.01), and the serum ALP had a trend of increasing too (P>0.05).Comparing with the model control group, the AST level was significantlydeclined (P<0.01) in both medicated groups after 7-week administration(FIG. 17). Comparing with the model control group, the ALT level wassignificantly declined (P<0.01) in both medicated groups after 7-weekadministration (FIG. 18).

General Observation of Liver Tissue, Liver Index and Fat Index:

In the model control group, it was observed that the liver volume of thehamsters increased obviously and the liver surface was greasy. The colorof the livers was also abnormal, which was grayish yellow or grayishwhite. The liver shape became blunt. The lipid deposition could beclearly observed. Moreover, both the liver weight and liver indexsignificantly increased (P<0.01).

Comparing with the model control group, there was no significant changeobserved in the body weight of either low-dose group or high-dose group(P>0.05). However, the liver weight of both the medicated groupssignificantly decreased (P<0.01). The detailed results were shown inFIG. 19.

Comparing with the model control group, the color of livers was improvedin both medicated groups. Especially in high-dose group the color oflivers was ruddy, which was similar to the case of normal control group.The detailed results were shown in FIG. 20.

Based on the results of pathological observation, the content of TC andTG in liver, the inflammation score of liver tissue and the positivearea for oil red significantly increased in the model control groupcomparing with those of the normal control group (P<0.01). Comparingwith the model control group, the content of TC and TG, the inflammationscore of liver tissue and the positive area for oil red significantlydecreased in both medicated group (P<0.01). The detailed results wereshown in FIG. 21-22.

Above experimental results indicated that BUDCA could significantlydecrease the level of TG, TC, LDL-c in serum, and could reduce theTC/HDL-c and the ambulatory arterial stiffness index (AI). It couldreduce the risk of atherosclerosis. It could significantly reduce thefatty deposits and improve the inflammation in liver. The effects ofBUDCA were relatively dose-dependent. And BUDCA would be a potentialcandidate to be applied in the treatment or prevention of NAFLD/NASH andhyperlipidemia.

Example 7. Synthesis Schemes of Exemplary Berberine Salts

A solution of ursolic acid (0.9-1.5 eq.) in methanol was treated with asolution of sodium bicarbonate (0.9-1.5 eq.) in water. The solution wasstirred for 30 minutes at room temperature, and then added dropwise to asolution of berberine chloride (1.0 eq.) in water. A yellow solidprecipitated immediately upon the addition. The mixture was stirred for1 hour, and then cooled to room temperature. A yellow solid was obtainedby filtration with the yield of 30% (NMR is shown in FIG. 15).

Example 8. Animal Models to Determine the Pharmacological Effects ofBerberine Salts

(1) Testing for Anti-Diabetes Activities

Healthy male Sprague-Dawley rats, 8 weeks of age, were placed in a roomwith controlled lighting (12 hours light/dark cycle) and regulatedtemperature (18° C.-25° C.) and humidity. All rats were fed with regularchow (protein 21%, carbohydrate 55%, fat 6%, and total energy 15.36KJ/g) for 1 week to be adapted for the environment. Six rats wererandomly selected as normal control group (NC), which were fed regularchow diet throughout the study. The remaining rats were fed withhigh-fat diet (protein 16%, carbohydrate 38%, fat 46%, and total energy20.54 KJ/g). After high-fat diet feeding for 8 weeks, diabetes wasinduced by a single intraperitoneal injection of freshly preparedstreptozotocin (STZ, 30 or 50 mg/Kg body weight) (Sigma, St. Louis, Mo.,USA) in citrate buffer (pH 4.5) to overnight fasted rats. After 2 wks ofSTZ administration, animals with fasting blood glucose levels >11.1 mMwere selected for the study, and randomized in the following groups:vehicle (water), low dose, mid dose and high dose of berberine saltsrespectively by intragastric administration once daily for 28consecutive days. The fasting glucose, insulin, total cholesterol,LDL-c, HDL-c and triglycerides levels of all animals were recorded onthe day before first dosing (day 0) and day 7, 14, 21 and 28 days ofdosing.

(2) Testing for Anti-Diabetic Complication (Diabetic Nephropathy)Activities

Five-week-old male Sprague-Dawley rats, weighing 120 to 130 g, were keptin wire-bottomed cages and exposed to a 12/12-h light/dark cycle. Theroom temperature was maintained at approximately 25° C. with relativeconstant humidity. They were allowed free access to regular laboratorypellet chow and water. After 1 week of adaptation, the rats underwentresection of one-half of the left kidney first, following by totalexcision of the right kidney 10 days after. Thereafter, they wereinjected intraperitoneally with STZ (25 mg/Kg body weight) in citratebuffer, pH 4.5. The blood glucose and urea nitrogen levels weredetermined after recovery from the injection, and the rats were dividedinto four groups (a control and three treatment groups), avoiding anyintergroup differences in these blood indices. A normal group of ratsthat underwent sham operation was also included. Each experimental groupcontained 10 rats. Whereas the 50-day experiment was performed, thenormal and control groups received water. The other three groupsreceived berberine salts at low, mid and high dose via intragastricgavage respectively. At the end of this experiment, 24-h urine sampleswere collected using metabolic cages, and blood samples were obtainedvia cardiac puncture. The serum was immediately separated from the bloodsamples by centrifugation. After renal perfusion through the renalartery with ice-cold physiological saline, the remaining kidney wasremoved from each rat, and one part of the tissue was immersed informalin for histological examination. The other part was frozen at −80°C. until analysis. Serum levels of glucose, total protein, albumin,total cholesterol, triglyceride, urea nitrogen, and creatinine wereexamined using commercial reagents.

(3) Testing for Anti-Dyslipidemia and/or Anti-Obesity Activities

The diet induced obesity (DIO) mice were established with feeding ahigh-fat diet (40 Kcal % fat) from 4 weeks of age of healthy NIH mice.Mice were housed three per group in polycarbonate cages maintained atnormal temperature (22±4° C.) with normal humidity and exposed to12/12-h light/dark cycle. After high-fat diet for 2 weeks, mice wereweighed and randomized into groups of 10 mice each: control groups, low,mid and high dose of berberine salt by intragastric gavage once dailyfor a total of 4 weeks with the high-fat diet throughout the treatment.And six normal mice were included as normal group with regular chowdiet. The food and water intake, body weight and non-fasting glucosewere tested for all animals on the day before first dosing (day 0) anddosing of day 7, 14, 21 and 28. The 6-h fasting glucose, insulin, totalcholesterol, LDL-c, HDL-c, triglyceride were tested for all animals onthe day before first dosing (day 0) and dosing of day 28. The oralglucose tolerance test (OGTT) was test after 12-h fasting on day 28.After OGTT test, all animals were sacrificed and the pancreas, liver,kidney, and fat were weighed and collected for histology analysis.

(4) Testing for Efficacy in Skeletal Muscle Atrophy Model

Thirty-two male Sprague-Dawley rats (age 8 weeks) were housedindividually at 25±1° C. with light from 8:00-20:00 and free access towater and regular commercial rat chow. After 1 week of acclimatization,rats were randomized into 4 groups. The control group (n=8) was injectedwith 2 mL/Kg/day of saline and the other three groups were injected with2 mg/Kg/day prednisolone, a glucocorticoid purchased from SIGMA-Aldrich(MO, USA). The three glucocorticoid-injected groups were treated withwater, low dose or high dose of berberine salts via intragastric gavagerespectively (n=8 per group) for a total of 4 weeks. The food and waterintake, body weight and glucose were tested for all animals on the daybefore first dosing (day 0) and dosing of day 7, 14, 21 and 28. At theend of the experiment, the rats were sacrificed by decapitation afterovernight fasting. Blood was collected and centrifuged at 3000 rpm for15 min to obtain serum. The serum was stored at −20° C. The liver, heartand skeletal muscles (soleus, plantaris, gastrocumemius, tibialisanterior and extensor digitorum longus) were quickly removed, weighedand stored at −80° C. until the analysis was performed.

(5) Testing for Efficacy in Attenuating NAFLD

Sixty-six healthy female Sprague-Dawley rats were randomized into twogroups: high fat diet group (n=56, fed with high-fat diet) and normalgroup (n=10, fed with regular diet). At the end of 12th week, 6 ratsfrom the high-fat diet group were randomly selected for hepatichistopathology examination and NAFLD rat model was confirmed to besuccessfully established. The remaining 50 model rats were subdividedinto 5 equal subgroups: low, mid and high dose of berberine salts viaintragastric gavage, vehicle control group and recovery group. Rats invehicle control group were fed with water by gavage. 20 weeks later, allrats were anesthetized by 3% pentobarbital sodium throughintraperitoneal injection. Plasma insulin and TG, TC, LDL-c, AST and ALTcontent in serum were determined. Upon sacrifice, liver tissues wereharvested for histopathology examination.

(6) Testing for Efficacy in Attenuating NASH

Male Sprague-Dawley rats, weighing 160-170 g and six weeks of age wereused in this study. They were housed in a temperature-controlled room(22±1° C.) with normal humidity and a 12 h light/dark cycle.

The rats were fed either standard chow (control group, n=8) orcholine-deficient high-fat (CDHF) diets through the experiment period of10 weeks. Fatty liver was induced by the feeding CDHF for 4 weeks. Inthe fifth week, rats on CDHF were randomized into six groups. The CDHFgroup (n=8) was fed CDHF diet only; the NASH group (n=8) rats were fedwith CDHF diet, followed by i.p. injections of sodium nitrite (NaNO₂),an oxidant, 50 mg/Kg/day (Nacalai Tesque Inc., Kyoto, Japan) daily toinduce methemoglobinemia (intermittent hypoxia stress) starting from the5th week of CDHF for 6 weeks; NASH plus low, mid, and high dose ofberberine salts (n=8 per group) via intragastric gavage concurrentlyduring the period of nitrite injection.

At the end of the 10-week experimental period, animals were sacrificedby anesthetizing with diethyl ether. Blood samples were collected byvena cava inferior puncture with syringe containing heparin, and wholebody perfusion was performed to left ventricular with 0.1 M potassiumcontaining 5 mM benzamidine before obtaining tissue samples. Plasma wasobtained by centrifugation at 1,000×g for 10 min at 4° C., and used forbiochemical analysis. Plasma alanine aminotransferase (ALT) andaspartate aminotransferase (AST) were determined with commercial kits.

Fresh liver was used for liver fractionation and for observation oflipid peroxidation, and a portion for histopathological observation wasimmersed in 10% formalin for 3 days and then embedded in paraffin. Andrest of liver was flash frozen by liquid nitrogen, stored in −80° C. forfurther analysis.

(7) Testing for Anti-Atherosclerosis Activities

The Atherosclerosis (AS) mice are established with feeding a compositionof 15% lard, 4.5% cholesterol diet from 4 weeks of age of healthy C57/BL6J mice. Mice are housed three per group in polycarbonate cagesmaintained at normal temperature (22±4° C.) with normal humidity andexposed to 12/12-h light/dark cycle.

After high-fat diet for 16 weeks, the histopathology of heart of 3-5mice from the model group is conducted to evaluate the modelestablishment. The atherosclerostic lesions in predisposedcholesterol-fed mice are most pronounced in the ascending aorta at theattachment of aortic valves to the sinus wall. In the control animals,there is a single layer of endothelial cell overlying a thin layer ofconnective tissue and elastic. No lipid droplets are seen. The modelmice are weighed and randomized into groups of 10 mice each: controlgroups, low, mid and high dose of berberine salt by intragastric gavageonce daily for a total of 8 weeks with the high-fat diet throughout thetreatment. And six normal mice are included as normal group with regularchow diet. The food and water intake and body weight are tested for allanimals on the day before first dosing (day 0) and dosing of day 14, 28,42 and 56. The total cholesterol, LDL-c, HDL-c, triglyceride are testedfor all animals on the day before first dosing (day 0) and dosing of day56. The all animals are sacrificed and the aorta, heart, liver, and fatare weighed and collected for histology analysis.

(8) Testing for Heart Failure Treatment

The efficacy of berberine salt for heart failure is evaluated with a ratmodel of dilated cardiomyopathy induced by Adriamycin. Adriamycin isinjected into male Wistar rats by intraperitoneal at the dose of 2 mg/Kgper 3 days for 5 times, then at a dose of 2 mg/Kg per 7 days for 5 timesto establish the heart failure model. Vehicle model group are injectedwith 0.9% saline using same methods. Rats are housed three per group inpolycarbonate cages maintained at normal temperature (22±4° C.) withnormal humidity and exposed to 12/12-h light/dark cycle with regularfeed. Four rats are randomly picked up to evaluate the heart functionwith transthoracic echocardiography and myocardium morphology withelectronmicroscope at the end of week 10. The parameters of LV enddiastolic diameter (LVEDD) and LV end systolic diameter (LVESD), LVejection fraction (EF) and LV faction shortening (FS) are shown that theheart failure of dilated cardiomyopathy type is established.

The rats are weighed and randomized into groups of 6 rats each: controlgroups, low, mid and high dose of berberine salt by intragastric gavageonce daily for a total of 8 weeks treatment. And six vehicle model ratsare included as normal group. The food and water intake and body weightare tested for all animals on the day before first dosing (day 0) anddosing of day 14, 28, 42 and 56. The parameters of LV end diastolicdiameter (LVEDD) and LV end systolic diameter (LVESD), LV ejectionfraction (EF) and LV faction shortening (FS) are tested on the day 56.After test, all animals are sacrificed and the heart, liver and kidneyare weighed and collected for histology analysis.

(9) Testing for Neurodegenerative Diseases Treatment

The efficacy of berberine salt for Parkinson disease is evaluated with aC57/BL6 mice induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine(MPTP). The mice are housed three per group in polycarbonate cagesmaintained at normal temperature (22±4° C.) with normal humidity andexposed to 12/12-h light/dark cycle with regular feed. 8-week old miceare intraperitoneally injected with MPTP at dose of 20 mg/kg/day for 7consecutive days, while the same volume of saline is injected in vehiclemodel rats with same method. The mice are weighed and randomized intogroups of 6 rats each: control groups, low, mid and high dose ofberberine salt by intragastric gavage once daily for a total of 8 weekstreatment. And six vehicle model rats are included as normal group.Injection of MPTP induced dopaminergic neuronal death in the substantianigra and fiber loss in the striatum, which results in impaired motorbalance and coordination, as assessed by the beam walking test. Bycontrast, treatment with berberine enhances motor balance andcoordination by preventing dopaminergic neuronal damage. Treatment withberberine also improves short-term memory by inhibiting apoptosis in thehippocampus.

In this specification and the appended claims, the singular forms “a,”“an,” and “the” include plural reference, unless the context clearlydictates otherwise.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art. Although any methods and materials similar or equivalent tothose described herein can also be used in the practice or testing ofthe present disclosure, the preferred methods and materials are nowdescribed. Methods recited herein may be carried out in any order thatis logically possible, in addition to a particular order disclosed.

INCORPORATION BY REFERENCE

References and citations to other documents, such as patents, patentapplications, patent publications, journals, books, papers, webcontents, have been made in this disclosure. All such documents arehereby incorporated herein by reference in their entirety for allpurposes. Any material, or portion thereof, that is said to beincorporated by reference herein, but which conflicts with existingdefinitions, statements, or other disclosure material explicitly setforth herein is only incorporated to the extent that no conflict arisesbetween that incorporated material and the present disclosure material.In the event of a conflict, the conflict is to be resolved in favor ofthe present disclosure as the preferred disclosure.

EQUIVALENTS

The representative examples disclosed herein are intended to helpillustrate the invention, and are not intended to, nor should they beconstrued to, limit the scope of the invention. Indeed, variousmodifications of the invention and many further embodiments thereof, inaddition to those shown and described herein, will become apparent tothose skilled in the art from the full contents of this document,including the examples which follow and the references to the scientificand patent literature cited herein. The following examples containimportant additional information, exemplification and guidance that canbe adapted to the practice of this invention in its various embodimentsand equivalents thereof.

What is claimed is:
 1. An acid-base addition salt in substantially pureform, having the formula of:(X⁺)_(m)(U⁻)_(n)  (I) wherein (a) U⁻ is an anionic moiety ofursodeoxycholic acid; (b) X⁺ is a cationic moiety of berberine; and (c)each of m and n is
 1. 2. A pharmaceutical composition comprising anacid-base addition salt having the formula of:(X⁺)_(m)(U⁻)_(n)  (I) wherein (a) U⁻ is an anionic moiety ofursodeoxycholic acid; (b) X⁺ is a cationic moiety of berberine; and (c)each of m and n is 1, wherein the acid-base addition salt has a purityequal to or greater than 95%, and a pharmaceutically acceptableexcipient, carrier, or diluent.
 3. The pharmaceutical composition ofclaim 2, consisting of: the acid-base addition salt having a purityequal to or greater than 95%, and a pharmaceutically acceptableexcipient, carrier, or diluent.